HK1251618B - Methods of detecting influenza - Google Patents

Description

Methods for detecting influenza

1. Field of the Invention

Compositions and methods for detecting influenza are provided. In particular, influenza markers and panels of markers for use in influenza detection are provided.

2. Background

Influenza (or flu) is a contagious viral infection of the respiratory tract. Flu is primarily spread through the air (i.e., coughing or sneezing); peak transmission often occurs during the winter months. Symptoms typically include fever, chills, headache, myalgia, malaise, cough, and sinus congestion. Gastrointestinal symptoms (i.e., nausea, vomiting, or diarrhea) may also occur, primarily in children but less commonly in adults. Symptoms typically appear within two days of exposure to the infection. Pneumonia can develop as a complication of influenza infection, causing increased morbidity and mortality in children, the elderly, and those with immunodeficiency. Influenza viruses are divided into type A (A), type B (B), and type C (C), with the first two causing the most human infections. Type A is the most common type of influenza virus in humans and typically causes seasonal influenza epidemics and occasionally pandemics. Type A viruses can also infect animals such as birds, pigs, and horses. Infections with type B viruses are typically limited to humans and rarely cause epidemics. Influenza A viruses are further divided into subtypes based on two surface proteins: hemagglutinin (H) and neuraminidase (N). Seasonal influenza is typically caused by subtypes H1, H2, H3, as well as N1 and N2. In addition to seasonal influenza, a new H1N1 strain was identified in humans in the United States in early 2009.

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family consisting of two strains (subgroups A and B), is also the cause of an infectious disease that primarily afflicts infants and the elderly who are immunocompromised (for example, those with chronic lung or heart disease or undergoing treatment for illnesses that weaken their immune systems). The virus can survive for hours on work surfaces and toys and cause upper respiratory tract infections (such as colds) as well as lower respiratory tract infections (manifesting as bronchitis and pneumonia). By age two, most children have been infected with RSV, but both children and adults can be reinfected due to their limited immunity. Symptoms typically develop four to six days after infection. The illness is usually self-limiting, lasting about one to two weeks in infants. In adults, the infection lasts about five days and presents with symptoms consistent with a cold, such as rhinorrhea, fatigue, headache, and fever. RSV season overlaps with flu season to some extent, as infections begin to rise during the fall and continue until early spring. However, RSV infections also occur at other times of the year, although less frequently.

Active surveillance programs combined with infection control alerts are an important component in preventing the spread of influenza and RSV. The use of assays that provide rapid results to identify patients infected with these seasonal infections is also an important factor in effective control, proper selection of treatment, and prevention of large outbreaks.

The genome of influenza virus comprises eight 0.9-2.3kb RNA segments, which together span about 13.5kb and encode 11 proteins. These 8 segments, named PB2, PB1, PA, HA, NP, NA, MP and NS, are under continuous selection pressure, which results in rapid sequence changes (antigenic drift). In addition to the changes in sequence level, influenza A also has the ability (antigenic shift) of exchanging the entire segment with other influenza A viruses. This process results in the emergence of epidemic influenza strains (i.e., influenza A H1N1pdm09, swine source (swing origin) H3N2).

Two proteins, hemagglutinin (HA) and neuraminidase (NA), determine the subtype of influenza A viruses (H and N, respectively). There are 16 H subtypes and 9 N subtypes. The H1N1 and H3N2 subtypes cause the vast majority of influenza infections in humans. Influenza B viruses have a similar RNA segment structure; however, influenza B viruses do not have subtypes.

This continuous antigenic drift and antigenic shift makes it difficult to maintain influenza detection assays between seasons.There remains a need for robust influenza detection assays that remain accurate even when the influenza genome undergoes genetic drift.

3. Overview

In some embodiments, methods of detecting the presence or absence of influenza in a sample from a subject are provided. In some embodiments, the methods include detecting the presence or absence of at least one influenza gene selected from acidic polymerase (PA) and basic polymerase 2 (PB2) in the sample.

In some embodiments, a method of determining whether a subject has influenza is provided. In some embodiments, the method comprises detecting the presence or absence of at least one influenza gene selected from the group consisting of an acidic polymerase (PA) gene and a basic polymerase 2 (PB2) gene in a sample from the subject.

In some embodiments, the method comprises detecting the presence or absence of a PA gene. In some embodiments, the method comprises detecting the presence or absence of a PB2 gene. In some embodiments, the method comprises detecting the presence or absence of both a PA gene and a PB2 gene. In some embodiments, the PA gene and/or the PB2 gene are influenza A genes. In some embodiments, the sequence of the PA gene is at least 95% identical to that of SEQ ID NO: 2. In some embodiments, the sequence of the PB2 gene is at least 95% identical to that of SEQ ID NO: 1.

In some embodiments, the method further comprises detecting the presence or absence of at least one influenza matrix protein (MP) gene. In some embodiments, the method comprises detecting the presence or absence of an influenza A MP gene. In some embodiments, the method comprises detecting the presence or absence of an influenza B MP gene. In some embodiments, the method comprises detecting the presence or absence of an influenza A MP gene and an influenza B MP gene. In some embodiments, the method comprises detecting the presence or absence of an avian influenza MP gene. In some embodiments, the avian influenza MP gene is of the hemagglutinin (H) 5 or H7 subtype. In some embodiments, the sequence of the influenza A MP gene is at least 95% identical to that of SEQ ID NO: 3 or 4. In some embodiments, the sequence of the influenza B MP gene is at least 95% identical to that of SEQ ID NO: 6.

In some embodiments, the method further comprises detecting the presence or absence of at least one influenza nonstructural (NS) gene. In some embodiments, the method further comprises detecting the presence or absence of an influenza B NS gene. In some embodiments, the sequence of the influenza B NS gene is at least 95% identical to the sequence of SEQ ID NO: 7.

In some embodiments, the method further comprises detecting the presence or absence of at least one influenza hemagglutinin (HA) gene. In some embodiments, the method further comprises detecting the presence or absence of an influenza A HA gene. In some embodiments, the method further comprises detecting the presence or absence of an avian influenza HA gene. In some embodiments, the avian influenza is of the H7 subtype. In some embodiments, the sequence of the influenza HA gene is at least 95% identical to the sequence of SEQ ID NO: 5.

In some embodiments, the method comprises detecting the presence or absence of an influenza A PA gene, an influenza A PB2 gene, an influenza A MP gene, an avian influenza MP gene, and an avian influenza HA gene. In some embodiments, the sequence of the influenza A PA gene is at least 95% identical to SEQ ID NO: 2, the sequence of the influenza A PB2 gene is at least 95% identical to SEQ ID NO: 1, the sequence of the influenza A MP gene is at least 95% identical to SEQ ID NO: 3, the sequence of the avian influenza MP gene is at least 95% identical to SEQ ID NO: 4, and the sequence of the avian influenza HA gene is at least 95% identical to SEQ ID NO: 5.

In some embodiments, the method comprises detecting the presence or absence of an influenza B MP gene and an influenza B NS gene. In some embodiments, the sequence of the influenza B MP gene is at least 95% identical to SEQ ID NO: 6 and the sequence of the influenza B NS gene is at least 95% identical to SEQ ID NO: 7.

In some embodiments, detecting the presence of any of the influenza genes indicates the presence of influenza in the sample. In some embodiments, the method distinguishes between influenza A and influenza B. In some embodiments, the method does not distinguish between influenza A and influenza B.

In some embodiments, the method comprises detecting the presence or absence of respiratory syncytial virus (RSV) in a sample from a subject. In some embodiments, the method comprises detecting the presence or absence of RSV A. In some embodiments, the method comprises detecting the presence or absence of RSV B. In some embodiments, the method comprises detecting the presence or absence of RSV A and RSV B.

In some embodiments, wherein the subject has more than one flu symptom.In some embodiments, the subject has more than one symptom selected from fever, chills, cough, sore throat, runny nose, nasal congestion, myalgia, headache, fatigue, vomiting and diarrhea.

In some embodiments, the method includes detecting an exogenous control. In some embodiments, the exogenous control is a sample processing control. In some embodiments, the exogenous control comprises an RNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is RNA.

In some embodiments, the method comprises PCR. In some embodiments, the method comprises quantitative PCR. In some embodiments, the PCR reaction takes less than 2 hours from the initial denaturation step to the final extension step.

In some embodiments, the method comprises contacting the nucleic acid from the sample with a first primer pair for detecting the influenza PA gene. In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 20, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 21. In some embodiments, the first primer has the sequence of SEQ ID NO: 20 and the second primer has the sequence of SEQ ID NO: 21.

In some embodiments, the method comprises contacting the nucleic acid from the sample with a second primer pair for detecting the influenza PB2 gene. In some embodiments, the second primer pair comprises a third primer and a fourth primer, wherein the third primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1, and wherein the fourth primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the second primer pair comprises a third primer and a fourth primer, wherein the third primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 17, and wherein the fourth primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 18. In some embodiments, the third primer has the sequence of SEQ ID NO: 17 and the fourth primer has the sequence of SEQ ID NO: 18.

In some embodiments, the method comprises contacting nucleic acid from the sample with at least one additional primer pair, wherein each additional primer pair is for detecting a different influenza gene selected from the group consisting of an influenza A MP gene, an avian influenza MP gene, and an avian influenza HA gene. In some embodiments, each additional primer pair comprises a fifth primer and a sixth primer, the fifth primer and the sixth primer being independently selected from: (a) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 3, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 3; (b) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: NO:4, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4; (c) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:5, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:5. NO: 5; (d) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 23; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 23. NO: 24; (e) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 26; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 27. NO:27; and (f) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:29; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:30.

In some embodiments, the method comprises contacting nucleic acid from the sample with at least one additional primer pair, wherein each additional primer pair is for detecting a different influenza gene selected from an influenza B MP gene and an influenza B NS gene. In some embodiments, each additional primer pair comprises a seventh primer and an eighth primer, the seventh primer and the eighth primer being independently selected from: (a) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6; (b) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: NO:7, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:7; (c) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:32, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:33; NO:33; and (d) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:35, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:36.

In some embodiments, the method comprises contacting nucleic acid from the sample with at least one additional primer pair, wherein each additional primer pair is for detecting RSV A or RSV B. In some embodiments, each additional primer pair comprises a ninth primer and a tenth primer, the ninth primer and the tenth primer being independently selected from: (a) a ninth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 38, and a tenth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 39; and (b) a ninth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: NO:41, and a tenth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:41.

In some embodiments, the method comprises contacting nucleic acid from the sample with primer pairs for detecting influenza A PA gene, influenza A PB2 gene, influenza A MP gene, avian influenza MP gene, and avian influenza HA gene. In some embodiments, the method further comprises contacting nucleic acid from the sample with primer pairs for detecting RSV A and RSV B. In some embodiments, the method comprises contacting nucleic acid from the sample with a control primer pair for detecting an exogenous control. In some embodiments, each primer pair produces an amplicon that is 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, or 50 to 150 nucleotides long.

In some embodiments, the method comprises forming an amplicon from each primer pair in the presence of a target for the primer pair. In some embodiments, the method comprises forming at least one amplicon selected from the group consisting of an influenza A PA amplicon and an influenza A PB2 amplicon. In some embodiments, the influenza A PA amplicon has a sequence of SEQ ID NO: 9 and the influenza A PB2 amplicon has a sequence of SEQ ID NO: 8.

In some embodiments, the method comprises forming at least one amplicon selected from the group consisting of an influenza A MP amplicon, an avian influenza MP amplicon, and an avian influenza HA amplicon. In some embodiments, the influenza A MP amplicon has a sequence of SEQ ID NO: 10, the avian influenza MP amplicon has a sequence of SEQ ID NO: 11, and the avian influenza HA amplicon has a sequence of SEQ ID NO: 12.

In some embodiments, the method further comprises forming an influenza B MP amplicon and/or an influenza B NS amplicon. In some embodiments, the influenza B MP amplicon has a sequence of SEQ ID NO: 13 and the influenza B NS amplicon has a sequence of SEQ ID NO: 14. In some embodiments, the method further comprises forming an RSV A amplicon and/or an RSV B amplicon. In some embodiments, the RSV A amplicon has a sequence of SEQ ID NO: 15 and the RSV B amplicon has a sequence of SEQ ID NO: 16.

In some embodiments, the method comprises contacting the amplicon with at least one probe selected from an influenza A PA probe and an influenza A PB2 probe. In some embodiments, the influenza PA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2, and the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the influenza PA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 19, and the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 22.

In some embodiments, the method comprises contacting the amplicon with at least one probe selected from the group consisting of an influenza A MP probe, an avian influenza MP probe, and an avian influenza HA probe. In some embodiments, the influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:3, and the avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4, and the avian influenza HA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:5. In some embodiments, the influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:25, and the avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:28, and the avian influenza HA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:31.

In some embodiments, the method comprises contacting the amplicon with at least one probe selected from an influenza B MP probe and an influenza B NS probe. In some embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 7. In some embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 34, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 37.

In some embodiments, the method comprises contacting the amplicon with at least one probe selected from an RSV A probe and an RSV B probe. In some embodiments, the RSV A probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 15, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:40, and the RSV B probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:43.

In some embodiments, each probe comprises a detectable label. In some embodiments, each probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the influenza A probe and the influenza B probe comprise a detectable label that is detectably distinguishable. In some embodiments, the influenza A probe and the influenza B probe comprise a detectable label that is not detectably distinguishable. In some embodiments, each probe consists of 15 to 30 nucleotides.

In some embodiments, the method includes forming an exogenous control amplicon. In some embodiments, the method includes contacting the exogenous control amplicon with a control probe capable of selectively hybridizing to the exogenous control amplicon.

In some embodiments, the method comprises detecting the presence or absence of at least one influenza A subtype and at least one influenza B subtype and an exogenous control in a single multiplex reaction. In some embodiments, the at least one influenza A subtype includes at least one avian influenza. In some embodiments, the method comprises detecting RSV A and/or RSV B in the same multiplex reaction.

In some embodiments, the sample is selected from the group consisting of a nasopharyngeal swab sample, a nasal aspirate sample, and a nasal wash sample.

In some embodiments, a composition is provided. In some embodiments, the composition comprises a first primer pair for detecting an influenza PA gene. In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 20, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 21. In some embodiments, the first primer has the sequence of SEQ ID NO: 20 and the second primer has the sequence of SEQ ID NO: 21.

In some embodiments, a composition is provided that comprises a second primer pair for detecting an influenza PB2 gene. In some embodiments, the second primer pair comprises a third primer and a fourth primer, wherein the third primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1, and wherein the fourth primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the second primer pair comprises a third primer and a fourth primer, wherein the third primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 17, and wherein the fourth primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 18. In some embodiments, the third primer has the sequence of SEQ ID NO: 17 and the fourth primer has the sequence of SEQ ID NO: 18.

In some embodiments, the composition comprises at least one additional primer pair, wherein each additional primer pair is for detecting a different influenza gene selected from the group consisting of an influenza A MP gene, an avian influenza MP gene, and an avian influenza HA gene. In some embodiments, each additional primer pair comprises a fifth primer and a sixth primer, the fifth primer and the sixth primer being independently selected from: (a) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 3, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 3; (b) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4; (c) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:5, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:5 NO: 5; (d) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 23; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 23. NO: 24; (e) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 26; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 27. NO:27; and (f) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:29; and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:30.

In some embodiments, the composition further comprises at least one additional primer pair, wherein each additional primer pair is for detecting a different influenza gene selected from the group consisting of an influenza B MP gene and an influenza B NS gene. In some embodiments, each additional primer pair comprises a seventh primer and an eighth primer, the seventh primer and the eighth primer being independently selected from: (a) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6; (b) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: NO:7, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:7; (c) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:32, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:33; NO:33; and (d) a seventh primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:35, and an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:36.

In some embodiments, the composition further comprises at least one additional primer pair, wherein each additional primer pair is for detecting RSV A or RSV B. In some embodiments, each additional primer pair comprises a ninth primer and a tenth primer, the ninth primer and the tenth primer being independently selected from: (a) a ninth primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 38, and a tenth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 39; and (b) a ninth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: NO:41, and a tenth primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:41.

In some embodiments, the composition comprises primer pairs for detecting the influenza A PA gene, the influenza A PB2 gene, the influenza A MP gene, the avian influenza MP gene, and the avian influenza HA gene. In some embodiments, the composition further comprises primer pairs for detecting RSV A and RSV B. In some embodiments, the composition further comprises primer pairs for detecting an exogenous control. In some embodiments, the exogenous control is a sample treatment control.

In some embodiments, the composition comprises at least one probe selected from an influenza A PA probe and an influenza A PB2 probe. In some embodiments, the influenza PA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 2, and the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the influenza PA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 19, and the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 22.

In some embodiments, the composition further comprises at least one probe selected from the group consisting of an influenza A MP probe, an avian influenza MP probe, and an avian influenza HA probe. In some embodiments, the influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:3, and the avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:4, and the avian influenza HA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:5. In some embodiments, the influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:25, and the avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:28, and the avian influenza HA probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:31.

In some embodiments, the composition further comprises at least one probe selected from the group consisting of an influenza B MP probe and an influenza B NS probe. In some embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 6, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 7. In some embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 34, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 37.

In some embodiments, the composition further comprises at least one probe selected from an RSV A probe and an RSV B probe. In some embodiments, the RSV A probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 15, and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:40, and the RSV B probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:43.

In some embodiments, the composition further comprises a probe for detecting an exogenous control.

In some embodiments, each probe comprises a detectable label. In some embodiments, each probe comprises a fluorescent dye and a quencher molecule. In some embodiments, each probe consists of 15 to 30 nucleotides.

In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is in solution. In some embodiments, the composition comprises nucleic acid from a sample from a subject tested for the presence or absence of influenza. In some embodiments, the sample is selected from a nasopharyngeal swab sample, a nasal aspirate sample, and a nasal wash sample.

In some embodiments, a kit is provided. In some embodiments, the kit comprises a composition as described herein. In some embodiments, the kit further comprises an exogenous control. In some embodiments, the exogenous control is RNA. In some embodiments, the kit comprises dNTPs and/or a thermostable polymerase. In some embodiments, the kit comprises a reverse transcriptase.

In some embodiments, oligonucleotides are provided consisting of a sequence selected from SEQ ID NOs: 17 to 43. In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the oligonucleotide comprises a detectable label. In some embodiments, the oligonucleotide comprises a fluorescent dye and a quencher molecule. In some embodiments, the oligonucleotide is a fluorescence resonance energy transfer (FRET) probe.

In some embodiments, a composition is provided, comprising a first primer consisting of the sequence of SEQ ID NO: 17 and a second primer consisting of the sequence of SEQ ID NO: 18, wherein the first primer and the second primer each comprise at least one modified nucleotide. In some embodiments, the composition comprises a probe consisting of the sequence of SEQ ID NO: 19, wherein the probe comprises at least one modified nucleotide and/or a detectable label.

In some embodiments, a composition is provided, comprising a first primer consisting of a sequence of SEQ ID NO: 20 and a second primer consisting of a sequence of SEQ ID NO: 21, wherein the first primer and the second primer each comprise at least one modified nucleotide. In some embodiments, the composition comprises a probe consisting of a sequence of SEQ ID NO: 22, wherein the probe comprises at least one modified nucleotide and/or a detectable label. In some embodiments, the probe is a fluorescence resonance energy transfer (FRET) probe. In some embodiments, the probe comprises at least one modified nucleotide. In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is in solution. In some embodiments, the composition comprises nucleic acid from a sample of a subject.

4. Detailed description

4.1 Definition

To facilitate understanding of the present invention, a number of terms and expressions are defined below:

As used herein, the terms "detect," "detecting," or "detection" may describe the general act of finding or identifying, or the specific observation of a detectably labeled composition.

As used herein, the term "detectably distinguishable" refers to a set of labels (eg, dyes) that can be detected and distinguished simultaneously.

As used herein, the terms "patient" and "subject" are used interchangeably to refer to a human. In some embodiments, the methods described herein can be used on samples from non-human animals.

As used herein, the terms "oligonucleotide," "polynucleotide," "nucleic acid molecule," and the like refer to molecules containing nucleic acids, including, but not limited to, DNA or RNA. The terms include sequences containing any known base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine , 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, -D-mannosyl queosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term "oligonucleotide" refers to a single-stranded polynucleotide having less than 500 nucleotides. In some embodiments, an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides long. An oligonucleotide can be named after its length, for example, an oligonucleotide of 24 residues can be referred to as a "24-aggressor."

As used herein, the term is "complementary" to a target RNA (or its target region), and the percentage of "complementarity" of a probe sequence to a target RNA sequence is the percentage of "identity" to the sequence of the target RNA or to the reverse complement of the sequence of the target RNA. In determining the degree of "complementarity" between the probe (or its region) and the target RNA (as disclosed herein) of the compositions herein, the degree of "complementarity" is expressed as the percentage of identity between the sequence of the probe (or its region) and the reverse complement of the sequence of the target RNA or its best aligned target RNA. The percentage is calculated by counting the number of identical aligned bases between the two sequences, dividing it by the total number of consecutive nucleotides in the probe, and multiplying it by 100. When the term "complementary" is used, the subject oligonucleotide is at least 90% complementary to the target molecule, unless otherwise stated. In some embodiments, the subject oligonucleotide is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

As used herein, a "primer" or "probe" refers to an oligonucleotide that comprises a region complementary to a sequence of at least 8 consecutive nucleotides of a target nucleic acid molecule, such as a DNA (e.g., a target gene) or an mRNA (or a DNA reverse transcribed from an mRNA). In some embodiments, a primer or probe comprises a region complementary to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of a target molecule. When a primer or probe comprises a region that is "complementary to at least x consecutive nucleotides of a target molecule," the primer or probe is at least 95% complementary to at least x consecutive nucleotides of the target molecule. In some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

The term "nucleic acid amplification" includes any means by which at least a portion of at least one target nucleic acid is replicated (typically in a template-dependent manner), including, but not limited to, a wide range of techniques for amplifying nucleic acid sequences (linearly or exponentially). Exemplary methods for performing the amplification step include polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), and the like, including multiple versions and combinations thereof, such as, but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), digital amplification, and the like. Descriptions of such techniques can be found in the following sources: Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb;4(1):41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the World Wide Web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acids Res. 20: 1691-96 (1992); Polstra et al., BMC Inf. Dis. 2: 18- (2002); Lage et al., Genome Res. 2003 Feb; 13(2): 294-307, and Landegren et al., Science 241: 1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.; 2(6): 542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb;12(1):21-7, U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

In some embodiments, amplification comprises at least one cycle of the following sequential steps: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; synthesizing nucleotides of at least one strand in a template-dependent manner using a polymerase; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycles may or may not be repeated. Amplification may include thermal cycling or may be performed isothermally.

Unless otherwise indicated, the term "hybridization" as used herein refers to "specific hybridization," which is the preferential binding, duplex formation, or hybridization of a nucleic acid molecule to a particular nucleotide sequence (in some embodiments, under stringent conditions). The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence and to other sequences to a lesser extent, or not hybridize at all. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization (e.g., as in array, Southern blot, or Northern blot hybridization) are sequence-dependent and are different under different environmental parameters. A comprehensive guide to nucleic acid hybridization is provided in, for example, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter 2, "Overview of principles of hybrizations and the strategy of nucleic acid probes assays," Elsevier, NY ("Tijssen"). Typically, highly stringent hybridization and wash conditions for filter hybridization are selected to be 5°C lower than the melting point ( _Tm ) for the specific sequence at a defined ionic strength and pH. _{The Tm} is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the _Tm for the specific probe. The dependence of hybridization stringency on buffer composition, temperature, and probe length is well known to those skilled in the art (see, e.g., Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY).

As used herein, "sample" includes various nasal samples, such as nasopharyngeal swab samples, nasal aspirate samples, nasal wash samples, and other types of human samples. In some embodiments, the nasal sample comprises a buffer, such as a preservative. Other non-limiting exemplary samples include nasal swabs, oropharyngeal swabs, laryngeal swabs, bronchoalveolar lavage samples, bronchial aspirates, bronchial washes, endotracheal aspirates, endotracheal washes, tracheal aspirates, nasal secretion samples, mucus samples, sputum samples, and lung tissue samples. In some embodiments, the sample comprises a buffer, such as a preservative.

As used herein, "endogenous control" refers to a portion naturally present in a sample to be used for detection. In some embodiments, the endogenous control is a "sample adequacy control" (SAC), which can be used to determine whether enough sample is used in the assay, or whether the sample contains enough biological material, such as cells. In some embodiments, the endogenous control is RNA (such as mRNA, tRNA, ribosomal RNA, etc.), such as human RNA. Non-limiting exemplary endogenous controls include ABLmRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPK1a mRNA. In some embodiments, such an endogenous control, such as a SAC, is selected so that it can be detected in the same manner as the target RNA, and in some embodiments, detected simultaneously with the target RNA.

As used herein, "exogenous control" refers to a part added to a sample or assay, such as a "sample processing control" (SPC). In some embodiments, an exogenous control is included therein together with an assay reagent. An exogenous control is typically selected to be expected not to be present in the sample to be detected, or to be present in the sample at very low levels, so that the amount of the part naturally present in the sample is not detectable as an exogenous control or is detected at a level far below the amount added to the sample. In some embodiments, an exogenous control comprises a nucleotide sequence that is expected not to be present in the sample type for detecting the target RNA. In some embodiments, an exogenous control comprises a nucleotide sequence that is known not to be present in the species from which the sample is collected. In some embodiments, an exogenous control comprises a nucleotide sequence from a species different from that from which the sample is collected. In some embodiments, an exogenous control comprises a nucleotide sequence that is known not to be present in any species. In some embodiments, an exogenous control is selected to be detected in the same manner as that for detecting the target RNA, and, in some embodiments, is detected simultaneously with the target RNA. In some embodiments, an exogenous control is RNA. In some such embodiments, the exogenous control is Armored, which comprises RNA packaged in a bacteriophage protective capsid. See, e.g., Walker Peach et al., Clin. Chem. 45: 12: 2079-2085 (1999).

In the sequences herein, "U" and "T" are used interchangeably so that the two letters indicate a uracil or thymine at that position. One skilled in the art will understand from the context and/or intended use whether a uracil or thymine is intended and/or should be used at that position in the sequence. For example, one skilled in the art will understand that natural RNA molecules typically include uracil, while natural DNA molecules typically include thymine. Thus, where an RNA sequence includes a "T," one skilled in the art will understand that the position in the natural RNA is likely to be a uracil.

In the present disclosure, "a sequence selected from..." includes both "a sequence selected from..." and "more than one sequence selected from..." Therefore, when "a sequence selected from..." is used, it is understood that one or more than one of the listed sequences can be selected.

In the present disclosure, a method comprising detecting "a set of influenza A (fluA) markers" includes detecting only the fluA markers of the set, and not detecting any other fluA markers. However, the method may include additional components or steps (such as for detecting fluB, respiratory syncytial virus (RSV), and/or endogenous and/or exogenous controls). Similarly, a method or composition comprising "a set of influenza A (fluA) marker primer pairs" and/or "a set of fluA marker probes" may include primer pairs and/or probes only for the fluA markers of the set, and not for any other fluA markers. However, the method or composition may include additional components, such as one or more fluB primer pairs, RSV primer pairs, endogenous control primer pairs, and/or exogenous control primer pairs.

4.2 Detection of influenza A

The inventors have developed a more sensitive assay for detecting influenza A. In some embodiments, the assay includes detecting influenza A alkaline polymerase 2 (PB2) gene and/or influenza A acidic polymerase (PA) gene. In some embodiments, the assay includes detecting PA and/or PB2 and, in addition, detecting influenza A matrix protein (MP) gene. This assay relies on polymerase chain reaction (PCR) and can be performed in a substantially automatic manner using a commercially available nucleic acid amplification system. Exemplary non-restrictive nucleic acid amplification systems that can be used to perform the method of the present invention include the PCR system, the Infinity system, and the Smartcycler System (Cepheid, Sunnyvale, CA). This assay can be completed within 3 hours, and in some embodiments, using an automatic system, for example, the system is completed within 2 hours.

4.2.1 General approach

Compositions and methods for detecting influenza A are provided. In some embodiments, the methods include detecting the influenza A PB2 gene and/or the PA gene. In some embodiments, the methods include detecting the influenza A PB2 gene and the PA gene. In some embodiments, the methods include detecting the influenza A PB2 gene and/or the PA gene and also detecting the influenza A MP gene. In some embodiments, the methods include detecting one or more of avian influenza (such as influenza A 2 and/or influenza A 3), Flu B, RSV A, and RSV B.

In some embodiments, a method for detecting influenza A in a subject comprises detecting the presence of the influenza A PB2 gene and/or the PA gene in a sample from the subject. In some embodiments, the method comprises detecting the influenza A PB2 gene and the PA gene in a sample from the subject. In some embodiments, the sample is selected from a nasopharyngeal swab sample, a nasal aspirate sample, and a nasal wash sample.

In some embodiments, the method of detecting influenza A further comprises detecting at least one endogenous control, such as a sample adequacy control (SAC). In some embodiments, the method of detecting influenza A further comprises detecting at least one exogenous control, such as a sample processing control (SPC). In some embodiments, the SPC is RNA.

In some embodiments, the method for detecting influenza A comprises detecting the influenza A PB2 gene and/or PA gene in a sample. In some embodiments, the method for detecting influenza A further comprises detecting a sample processing control (SPC), such as RNA.

In this disclosure, the terms "target RNA" and "target gene" are used interchangeably to refer to the influenza A PB2 gene and the influenza A PA gene, and also to other Flu and RSV genes, as well as exogenous and/or endogenous controls. Therefore, it is to be understood that when there is a discussion about a target gene, the discussion is specifically intended to include the influenza A PB2 gene and the influenza A PA gene, other Flu and RSV genes, any one or more endogenous controls (e.g., SAC), and any one or more exogenous controls (e.g., SPC).

In some embodiments, the presence of the influenza A PB2 gene and/or the influenza A PA gene is detected in a nasal sample. In some embodiments, the target gene is detected in a nasal aspirate sample or a nasal wash sample. In some embodiments, the target gene is detected in a sample to which a buffer (e.g., a preservative) has been added. In some embodiments, the presence of the influenza A PB2 gene and/or the influenza A PA gene is detected in a nasopharyngeal swab sample. In some embodiments, the target gene is detected in a nasopharyngeal swab sample placed in a buffer (e.g., a preservative).

In some embodiments, detection of the influenza A PB2 gene and/or the influenza A PA gene in a sample from a subject indicates the presence of influenza A in the subject. In some embodiments, detection of the influenza A PB2 gene and/or the influenza A PA gene in a sample from a subject indicates the presence of influenza A 1 in the subject. In some embodiments, detection is performed quantitatively. In other embodiments, detection is performed qualitatively. In some embodiments, detecting the target gene comprises forming a complex comprising a polynucleotide and a nucleic acid selected from the group consisting of the target gene, a cDNA reverse transcribed from the target gene, a DNA amplicon of the target gene, and a complement of the target gene. In some embodiments, detecting the target gene comprises RT-PCR. In some embodiments, detecting the target gene comprises quantitative RT-PCR or real-time RT-PCR. In some embodiments, a sample adequacy control (SAC) and/or a sample processing control (SPC) are detected in the same assay as the target gene. In some embodiments, if the influenza A PB2 gene or the influenza A PA gene is detected, influenza A is considered to be detected, even if the SPC is not detected in the assay. In some embodiments, influenza A is considered not detected only if SPC is detected in the assay if both the influenza A PB2 gene and the influenza A PA gene are not detected.

In some embodiments, the presence of the influenza A PB2 gene and/or the influenza A PA gene can be measured in samples collected from a subject at one or more times to monitor treatment of Flu in the subject. In some embodiments, this assay can be used as part of routine and/or preventive care for the subject. In some embodiments, this assay can be used seasonally as part of routine and/or preventive care for the subject. In some embodiments, this assay can be used as part of routine and/or preventive care for the subject who is at particular risk for influenza, such as immunocompromised and elderly subjects.

In some embodiments, the sample to be tested is a nasal aspirate sample or a nasal wash sample, or is derived from a nasal aspirate sample or a nasal wash sample. In some embodiments, a buffer (e.g., a preservative) is added to the nasal aspirate sample or the nasal wash sample. In some embodiments, the buffer is added to the nasal aspirate sample or the nasal wash sample within 5 minutes, 10 minutes, 30 minutes, 1 hour, or 2 hours of collecting the sample.

In some embodiments, the sample to be tested is a nasopharyngeal swab sample. In some embodiments, the swab is placed in a buffer. In some embodiments, the swab is immediately placed in a buffer. In some embodiments, the swab is placed in a buffer within 5 minutes, within 10 minutes, within 30 minutes, within 1 hour, or within 2 hours of collecting the sample.

In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of sample or buffered sample is used in the present method. In some embodiments, 0.1 ml to 1 ml of sample or buffered sample is used in the present method.

In some embodiments, the sample to be tested is another body fluid, such as saliva, a nasal swab, an oropharyngeal swab, a laryngeal swab, a bronchoalveolar lavage sample, a bronchial aspirate, a bronchial wash, an endotracheal aspirate, an endotracheal wash, a tracheal aspirate, a nasal secretion sample, a mucus sample, a sputum sample, a lung tissue sample, etc.

The clinical sample to be tested, in some embodiments, is fresh (i.e., not frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the sample is a tissue sample, such as a formalin-fixed wax-embedded sample. In some embodiments, the sample is a liquid cytology sample.

In some embodiments, the sample to be tested is obtained from an individual with one or more symptoms of influenza infection. Non-limiting exemplary symptoms of influenza include fever, chills, cough, sore throat, runny nose, nasal congestion, myalgia, headache, fatigue, vomiting, diarrhea, and any combination of those symptoms. In some embodiments, the sample to be tested is obtained from an individual previously diagnosed with influenza. In some such embodiments, the recurrence of influenza in the monitoring individual is performed.

In some embodiments, the methods described herein can be used for routine screening of healthy individuals without risk factors. In some embodiments, the methods described herein are used to screen asymptomatic individuals, for example, during routine or preventive care. In some embodiments, the methods described herein are used to screen women who are pregnant or attempting to become pregnant.

In some embodiments, the methods described herein can be used to assess the effectiveness of a treatment for an influenza infection in a patient.

In some embodiments, the use of the acidic polymerase (PA) gene and/or the alkaline polymerase 2 (PB2) gene for detecting influenza A is provided. In some embodiments, the use of the PA gene and/or the PB2 gene for detecting influenza A is provided. In some embodiments, the use of the PA gene and/or the PB2 gene, and optionally, one or more genes selected from the matrix protein (MP) gene and the nonstructural protein (NP) gene, for detecting influenza A is provided. In some embodiments, the use of the PA gene, the PB2 gene, the MP gene, and the NP gene for detecting influenza A and influenza B is provided.

In any of the embodiments described herein, the acidic polymerase (PA) gene and/or the basic polymerase 2 (PB2) gene can be detected in the same assay reaction as a sample processing control (SPC).

In some embodiments, a method for facilitating the detection of influenza A infection in a subject is provided. The method comprises detecting the presence or absence of the influenza A PB2 gene and/or the influenza A PA gene in a sample from the subject. In some embodiments, information about the presence or absence of the influenza A PB2 gene and/or the influenza A PA gene in a sample from the subject is communicated to a licensed physician. As used herein, "licensed physician" refers to an individual or entity that diagnoses and/or treats a patient, such as a hospital, clinic, doctor's office, doctor, nurse, or any of the aforementioned entities and individuals. In some embodiments, the detection of the presence or absence of the influenza A PB2 gene and/or the influenza A PA gene is performed in a laboratory that receives a sample from a subject from a licensed physician or a licensed physician's institution. The laboratory performs the detection by any method (including those described herein) and then communicates the results to the licensed physician. When it is provided to the licensed physician by any means, the results are "communicated" as used herein. In some embodiments, such communication may be oral or written, may be by telephone, in person, by email, by mail or other courier, or may be performed by directly storing the information in, for example, a database accessible to the practitioner, including databases not controlled by the practitioner. In some embodiments, the information is maintained in electronic form. In some embodiments, the information is stored in memory or other computer-readable media, such as RAM, ROM, EEPROM, flash memory, computer chips, digital video disks (DVDs), compact disks (CDs), hard disks (HDDs), magnetic tape, etc.

In some embodiments, a method for detecting influenza A is provided. In some embodiments, a method for diagnosing influenza A infection is provided. In some embodiments, the method includes obtaining a sample from a subject and providing the sample to a laboratory for detecting the influenza A PB2 gene and/or the influenza A PA gene in the sample. In some embodiments, the method further includes receiving a communication from the laboratory indicating the presence or absence of the influenza A PB2 gene and/or the influenza A PA gene in the sample. As used herein, a "laboratory" is any facility that detects a target gene in a sample by any method, including the methods described herein, and communicates the results to a licensed physician. In some embodiments, the laboratory is under the control of a licensed physician. In some embodiments, the laboratory is not under the control of a licensed physician.

When the laboratory communicates the results of detecting the presence or absence of the influenza A PB2 gene and/or the influenza A PA gene to the medical practitioner, in some embodiments, the laboratory indicates whether the influenza A PB2 gene and/or the influenza A PA gene were detected in the sample. In some embodiments, the laboratory indicates whether the sample contains influenza A by indicating, for example, "influenza positive" or "influenza negative" or "influenza present" or "influenza absent," etc.

As used herein, when a method relates to detecting influenza A, determining the presence of influenza A, monitoring influenza A, and/or diagnosing influenza A infection, the method includes activities in which the steps of the method are performed, but the result is negative for the presence of influenza A. That is, detecting, determining, monitoring, and diagnosing influenza A or influenza A infection include situations in which performing the method results in either a positive or negative result.

In some embodiments, at least one endogenous control (e.g., SAC) and/or at least one exogenous control (e.g., SPC) are detected simultaneously with the influenza A PB2 gene and/or the influenza A PA gene in a single reaction. In some embodiments, at least one exogenous control (e.g., SPC) is detected simultaneously with the influenza A PB2 gene and/or the influenza A PA gene in a single reaction.

In any of the embodiments described herein, the influenza A PB2 gene and/or the influenza A PA gene can be detected together with one or more additional influenza genes, including, but not limited to, influenza A 1 MP, influenza A 2 MP, influenza A 3 hemagglutinin (HA), and influenza B MP. In any of the embodiments described herein, the influenza A PB2 gene and/or the influenza A PA gene can be detected together with one or more additional influenza genes (such as those listed above), and RSV, such as RSV A and/or RSV B.

4.2.2 Exemplary Controls

In some embodiments, the assays described herein include detecting the influenza A PB2 gene and/or the influenza A PA gene and at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). In some such embodiments, if neither the influenza A PB2 gene nor the influenza A PA gene is detected in the sample, and no SAC is detected in the sample, the assay result is considered "invalid" because the sample may be insufficient. Without intending to be bound by any theory, an insufficient sample may be too dilute, contain too little cell material, contain assay inhibitors, etc. In some embodiments, failure to detect a SAC may indicate a failure of the assay reaction. In some embodiments, the endogenous control is RNA (such as mRNA, tRNA, ribosomal RNA, etc.). Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPK1a mRNA.

In some embodiments, the assays described herein include detecting the influenza A PB2 gene and/or the influenza A PA gene and at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). In some such embodiments, if the PB2 gene and/or the PA gene are not detected in the sample and the SPC is also not detected in the sample, the assay result is considered "invalid" because there may be errors in the sample processing, including but not limited to assay failure. Non-limiting exemplary errors in sample processing include insufficient sample processing, the presence of assay inhibitors, the presence of nucleases (such as RNases), damaged reagents, and the like. In some embodiments, an exogenous control (such as an SPC) is added to the sample. In some embodiments, an exogenous control (such as an SPC) is added during the assay, such as together with one or more buffers or reagents. In some embodiments, when the system is used, the SPC is included in the cartridge. In some embodiments, the exogenous control (such as an SPC) is Armored, which is protected by a phage coat.

In some embodiments, an endogenous control and/or an exogenous control are detected simultaneously in the same assay as the PA gene and/or the PB2 gene. In some embodiments, the assay includes reagents for simultaneously detecting the PA gene and/or the PB2 gene and the exogenous control in the same assay reaction. In some such embodiments, for example, the assay reaction includes a primer set for amplifying the PA gene and/or a primer set for amplifying the PB2 gene, as well as a primer set for amplifying the exogenous control, and a labeled probe (such as, for example, a probe) for detecting the amplified product.

4.2.3 Exemplary Sample Preparation

4.2.3.1 Exemplary Buffers

In some embodiments, a buffer is added to the sample. In some embodiments, the buffer is added within one hour, two hours, three hours, or six hours of the time the sample is collected. In some embodiments, the buffer is added to the sample within one hour, two hours, three hours, or six hours before the sample is analyzed using the methods described herein.

In some embodiments, the swab sample is placed in a buffer. In some embodiments, the swab sample is placed in a buffer within one hour, two hours, three hours, or six hours of the time the swab sample is collected. In some embodiments, the swab sample is placed in a buffer within one hour, two hours, three hours, or six hours before the sample is analyzed using the methods described herein.

Non-limiting exemplary commercial buffers include viral transport media provided with the Nasal Pharyngeal Collection Kit (Cepheid, Sunnyvale, CA); Universal Transport Medium (UTM ^™ , Copan, Murrieta, CA); Universal Viral Transport Medium (UVT, BD, Franklin Lakes, NJ); M4, M$RT, M5, and M6 (Thermo Scientific). Further non-limiting exemplary buffers include liquid Amies medium, PBS/0.5% BSA, PBS/0.5% gelatin, Bartel BirA Trans ^™ medium, EMEM, PBS, EMEM/1% BSA, sucrose phosphate, Trypticase ^™ soy broth (with or without 0.5% gelatin or 0.5% BSA), modified Stuart's medium, beef infusion broth (with or without 0.5% BSA), and saline.

4.2.3.2 Exemplary RNA Preparation

Target RNA can be prepared by any appropriate method. Total RNA can be isolated by any method, including, but not limited to, the protocol described in Wilkinson, M. (1988) Nucl. Acids Res. 16 (22): 10,933; and Wilkinson, M. (1988) Nucl. Acids Res. 16 (22): 10934, or by using commercially available kits or reagents, such as reagents (Invitrogen), total RNA extraction kit (iNtRON Biotechnology), total RNA purification kit (NorgenBiotek Corp.), RNAqueous ^™ (Ambion), MagMAX ^™ (Ambion), RecoverAll ^™ (Ambion), RNAeasy (Qiagen), etc.

In some embodiments, RNA levels are measured in samples in which RNA has not been first purified from cells. In some such embodiments, cells are subjected to a lysis step to release RNA. Non-limiting exemplary lysis methods include ultrasound (e.g., 2-15 seconds, 8-18 μm at 36 kHz); chemical lysis, e.g., using detergents; and various commercially available lysis reagents (e.g., RNAeasy lysis buffer, Qiagen). In some embodiments, RNA levels are measured in samples in which RNA has been isolated.

In some embodiments, the RNA is modified prior to detecting the target RNA. In some embodiments, all RNA in the sample is modified. In some embodiments, only the specific target RNA to be analyzed is modified, for example, in a sequence-specific manner. In some embodiments, the RNA is reverse transcribed. In some such embodiments, the RNA is reverse transcribed using MMLV reverse transcriptase. Non-limiting exemplary conditions for reverse transcribing RNA using MMLV reverse transcriptase include incubation at 40°C to 50°C for 5 to 20 minutes.

In some embodiments, the target RNA is reverse transcribed, and the target RNA is formed into a DNA complementary object. In some embodiments, the target RNA is detected by a complementary object rather than the target RNA itself (or a DNA copy of the RNA itself). Therefore, when the method discussed herein shows that the target RNA is detected, or when determining the level of the target RNA, this detection or determination can be carried out on the complementary object of the target RNA, rather than the target RNA itself (or except the RNA itself). In some embodiments, when the target RNA is detected by a complementary object rather than the target RNA, the polynucleotide used for detection is used, which is complementary to the complementary object of the target RNA. In some such embodiments, the polynucleotide used for detection comprises at least a portion identical with the target RNA sequence, although it can contain thymine to replace uracil, and/or comprises the nucleotide of other modifications.

4.2.4 Exemplary Analysis Methods

As described above, there is provided detection influenza, and optionally, a method for detecting respiratory syncytial virus (RSV).Methods described includes the presence of influenza A alkaline polymerase 2 (PB2) gene and/or acidic polymerase (PA) gene in the sample from the experimenter.In some embodiments, methods described also includes detecting and being selected from influenza A 1 matrix protein (MP) gene, influenza A 2 matrix protein (MP) gene, influenza A 3 hemagglutinin (HA) gene, influenza B non-structural protein (NS) gene, RSV A genome, or RSV B genome, and optionally, more than one other target gene in at least one exogenous control (such as SPC).In some embodiments, detecting and being selected from influenza A alkaline polymerase 2 (PB2) gene, acidic polymerase (PA) gene, influenza A 1 matrix protein (MP) gene, influenza A 2 matrix protein (MP) gene, influenza A 3 hemagglutinin (HA) gene, and influenza B non-structural protein (NS) gene more than one gene shows the presence of influenza, even if endogenous control and/or exogenous control are not detected in mensuration. In some embodiments, detection of RSV A or RSV B indicates the presence of RSV, even if the endogenous control and/or exogenous control is not detected in the assay. In some embodiments, if any of the influenza target genes (such as influenza A alkaline polymerase 2 (PB2) gene, acidic polymerase (PA) gene, influenza A 1 matrix protein (MP) gene, influenza A 2 matrix protein (MP) gene, influenza A 3 hemagglutinin (HA) gene, and influenza B nonstructural protein (NS) gene) are not detected, the test is considered negative for influenza only if the control is detected.

Any analytical step that can allow specific detection of a target gene can be used in the methods provided herein. Exemplary non-limiting analytical steps include, but are not limited to, nucleic acid amplification methods, PCR methods, isothermal amplification methods, and other analytical detection methods known to those skilled in the art.

In some embodiments, methods for detecting a target gene, such as an influenza A 1 matrix protein (MP) gene or an influenza A 2 matrix protein (MP) gene, include amplifying the gene and/or its complement. Such amplification can be achieved by any method. Exemplary methods include, but are not limited to, isothermal amplification, real-time RT-PCR, endpoint RT-PCR, and amplification using T7 polymerase from a T7 promoter annealed to DNA, such as provided by the SenseAmp Plus™ kit available in Implen, Germany.

When amplifying the target gene, in some embodiments, the amplicon of the target gene is formed. The amplicon can be single-stranded or double-stranded. In some embodiments, when the amplicon is single-stranded, the sequence of the amplicon is relevant to the target gene in the sense or antisense direction. In some embodiments, the amplicon of the target gene is detected rather than the target gene itself. Therefore, when the method discussed herein shows to detect the target gene, this detection can be carried out on the amplicon of the target gene (rather than the target gene itself, or in addition to the target gene itself). In some embodiments, when detecting the amplicon of the target gene rather than the target gene, the polynucleotide used for detection is complementary to the complement of the target gene. In some embodiments, when detecting the amplicon of the target gene rather than the target gene, the polynucleotide used for detection is complementary to the complement of the target gene. In some embodiments, when detecting the amplicon of the target gene rather than the target gene, the polynucleotide used for detection is complementary to the complement of the target gene. In addition, in some embodiments, the multiple polynucleotides used for detection can be used, and some polynucleotides can be complementary to the target gene and some polynucleotides can be complementary to the complement of the target gene.

In some embodiments, the method for detecting a target gene, such as the influenza A-1 matrix protein (MP) gene or the influenza A-2 matrix protein (MP) gene, comprises PCR, as described below. In some embodiments, detecting one or more target genes comprises real-time monitoring of the PCR reaction, which can be achieved by any method. Such methods include, but are not limited to, the use of molecular beacons, or Scorpion probes (i.e., energy transfer (ET) probes, such as FRET probes) and the use of intercalating dyes, such as SYBR Green, EvaGreen, Thiazole Orange, YO-PRO, TO-PRO, and the like.

Non-limiting exemplary conditions for amplifying cDNA reverse transcribed from target RNA are as follows. Exemplary cycles include initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes, followed by cycling, wherein the cycling is comprised of denaturation at 90°C to 100°C for 1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40 seconds. Further exemplary cycling includes 20 seconds at 94°C, followed by 1 second at 95°C, 3 cycles of 35 seconds at 62°C, 1 second at 95°C, 20 cycles of 20 seconds at 62°C, and 1 second at 95°C, 14 cycles of 35 seconds at 62°C. In some embodiments, the cyclic denaturation step is omitted for the first cycle after the initial denaturation step. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycle is performed at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 45 times. In some embodiments, a Taq with hot start functionality is used. In some embodiments, the amplification reaction occurs in the cartridge, and amplification of the target gene and the exogenous control occurs in the same reaction. In some embodiments, detection of the target gene occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from initial denaturation to final extension.

In some embodiments, the detection of the target gene includes forming a complex comprising a polynucleotide complementary to the target gene or its complement, and a nucleic acid selected from the target gene, the DNA amplicon of the target gene and the complement of the target gene. Therefore, in some embodiments, the polynucleotide forms a complex with the target gene. In some embodiments, the polynucleotide forms a complex with the complement of the target RNA (such as cDNA reverse transcribed from the target RNA). In some embodiments, the polynucleotide forms a complex with the DNA amplicon of the target gene. When the double-stranded DNA amplicon is part of a complex as used herein, the complex can include one or two chains of the DNA amplicon. Therefore, in some embodiments, the complex only includes one chain of the DNA amplicon. In some embodiments, the complex is a triplex and includes two chains of the polynucleotide and the DNA amplicon. In some embodiments, the complex is formed by hybridization between the polynucleotide and the target gene, the complement of the target gene, or the DNA amplicon of the target gene. Polynucleotides, in some embodiments, are primers or probes.

In some embodiments, the method includes detecting a complex. In some embodiments, the complex does not necessarily need to be associated when detected. That is, in some embodiments, a complex is formed, then the complex is dissociated or destroyed in some manner, and the components from the complex are detected. An example of such a system is an assay. In some embodiments, when the polynucleotide is a primer, the detection of the complex can include amplifying the target gene, the complement of the target gene, or a DNA amplicon of the target gene.

In some embodiments, the analytical method for detecting at least one target gene in the methods described herein includes real-time quantitative PCR. In some embodiments, the analytical method for detecting at least one target gene includes the use of a probe. The assay uses energy transfer ("ET"), such as fluorescence resonance energy transfer ("FRET"), to detect and quantitatively synthesize PCR products. Typically, the probe comprises a fluorescent dye molecule connected to the 5'-end and a quencher molecule connected to the 3'-end, so that the dye and the quencher are in close proximity, so that the quencher suppresses the fluorescent signal of the dye by FRET. When the polymerase replicates the chimeric amplicon template bound by the probe, the 5'-nuclease of the polymerase cuts the probe, uncoupling the dye from the quencher, thereby detecting the signal (such as fluorescence) of the dye. The signal (such as fluorescence) increases proportionally with each PCR cycle and the amount of the probe cut.

In some embodiments, if any signal is generated from the TaqMan probe during the PCR cycle, the target gene is considered to be detected. For example, in some embodiments, if the PCR comprises 40 cycles, if a signal is generated at any cycle during the amplification process, the target gene is considered to be present and detected. In some embodiments, if no signal is generated at the end of the PCR cycle, the target gene is considered to be absent and not detected.

In some embodiments, the quantitative determination of the real-time PCR results is performed by constructing a standard curve from nucleic acids of known concentrations and then extrapolating the quantitative information of the target gene of unknown concentration. In some embodiments, the nucleic acid used to generate the standard curve is DNA (e.g., an endogenous control, or an exogenous control). In some embodiments, the nucleic acid used to generate the standard curve is a purified double-stranded plasmid DNA or single-stranded DNA produced in vitro.

In some embodiments, in order for an assay to indicate the absence of influenza in a sample, the Ct value for an endogenous control (e.g., SAC) and/or an exogenous control (e.g., SPC) must be within a previously determined effective range. That is, in some embodiments, the absence of influenza cannot be confirmed unless a control is detected, indicating a successful assay. In some embodiments, the assay includes an exogenous control. The Ct value is inversely proportional to the amount of nucleic acid target in the sample.

In some embodiments, a threshold Ct (or "cutoff Ct") value for a target gene (including an endogenous control and/or an exogenous control) is previously determined (below which the gene is considered detected). In some embodiments, the threshold Ct is determined using substantially the same assay conditions and system (e.g., ) on which the sample will be tested.

In addition to assays, other real-time PCR chemistries used to detect and quantify PCR products in the methods described herein include, but are not limited to, molecular beacons, Scorpion probes, and intercalating dyes such as SYBR Green, EvaGreen, Thiazole Orange, YO-PRO, TO-PRO, etc., which are discussed below.

In various embodiments, real-time PCR detection is used to detect influenza target genes, and optionally, one or more RSV target genes, endogenous controls, and exogenous controls in a single multiplex reaction. In some multiplex embodiments, multiple probes are used, such as probes that are each specific for a different target. In some embodiments, each target gene-specific probe is spectrally distinguishable from the other probes used in the same multiplex reaction. Non-limiting exemplary seven-color multiplex systems are described, for example, in Lee et al., BioTechniques, 27:342-349.

In some embodiments, the quantitative use of real-time RT PCR products in conjunction with double-stranded DNA product dyes (such as SYBRGreen, EvaGreen, thiazole orange, YO-PRO, TO-PRO etc.) is completed. In some embodiments, determination is the QuantiTect SYBR Green PCR determination from Qiagen. In this determination, total RNA is first isolated from the sample. The total RNA is then polyadenylated at the 3'-end and reverse transcribed using a universal primer with poly-dT at the 5'-end. In some embodiments, a single reverse transcription reaction is sufficient to measure multiple target RNAs. Target RNA-specific primers and miScript Universal primers (which comprise poly-dT sequences at the 5'-end) are then used to complete real-time RT-PCR. SYBR Green dye non-specifically binds to double-stranded DNA and emits light when excited. In some embodiments, buffer conditions that promote highly specific annealing of primers to PCR templates (e.g., available from the QuantiTect SYBR Green PCR kit from Qiagen) can be used to avoid the formation of nonspecific DNA duplexes and primer dimers that would bind to SYBR Green and negatively affect quantification. Thus, as PCR product accumulates, the signal from SYBR Green increases, allowing quantification of specific products.

Real-time PCR is performed using any PCR instrument available in the art. Typically, instruments used for real-time PCR data collection and analysis include a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

In some embodiments, the detection and/or quantification of real-time PCR products are completed using dyes (such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc.) that bind double-stranded DNA products. In some embodiments, the analytical method for the methods described herein is (DNA-mediated annealing, selection, extension and connection) determination. In some embodiments, total RNA is isolated from the sample to be analyzed by any method. The total RNA can then be polyadenylated (>18 A residues are added to the 3'-end of the RNA in the reaction mixture). RNA is reverse transcribed using a biotin-labeled DNA primer, the primer comprising a sequence in the poly-dT region of the poly-dA tail comprising a PCR primer site and the sample RNA from 5' to 3' end. The biotinylated cDNA transcript obtained is then hybridized to a solid support by biotin-streptavidin interaction and contacted with more than one target RNA-specific polynucleotide. The target RNA-specific polynucleotide comprises the following from 5' to 3' end: a region comprising a PCR primer site, a region comprising an address sequence, and a target RNA-specific sequence.

In some embodiments, the target RNA-specific sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 consecutive nucleotides having a sequence that is identical or complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 consecutive nucleotides of a target RNA, an endogenous control RNA, or an exogenous control RNA.

After hybridization, the target RNA-specific polynucleotide is extended, and the extended product is then eluted from the immobilized cDNA array. A second PCR reaction using a fluorescent-labeled universal primer produces a fluorescent-labeled DNA comprising the target RNA-specific sequence. The PCR product of the label is then hybridized with a microbead array for detection and quantification.

In some embodiments, the analytical method used to detect and quantify the target gene in the methods described herein is a bead-based flow cytometric assay. See Lu J. et al. (2005) Nature 435: 834-838, which is incorporated herein by reference in its entirety. An example of a bead-based flow cytometric assay is the technology of Luminex, Inc. See www.luminexcorp.com/technology/index.html. In some embodiments, total RNA is isolated from a sample and subsequently labeled with biotin. The labeled RNA is then hybridized with a target RNA-specific capture probe covalently bound to a microbead (e.g., the FlexmiR ^™ product sold by Luminex, Inc. at www.luminexcorp.com/products/assays/index.html), each of which is labeled with two dyes having different fluorescence intensities. A streptavidin-bound reporter molecule (e.g., streptavidin-phycoerythrin, also known as "SAPE") is attached to the captured target RNA and the unique signal of each bead is read using flow cytometry. In some embodiments, the RNA sample is first polyadenylated and then labeled with a biotinylated 3DNA™ dendrimer (i.e., a multi-arm DNA having multiple biotin molecules bound thereto) using a bridging polynucleotide complementary to the 3'-end of the poly- ^dA tail of the sample RNA and to the 5'-end of the polynucleotide attached to the biotinylated dendrimer. Streptavidin-bound reporter molecules are then attached to the biotinylated dendrimer before analysis by flow cytometry. In some embodiments, the biotin-labeled RNA is first exposed to a SAPE, and then the RNA/SAPE complex is exposed to an anti-phycoerythrin antibody attached to a DNA dendrimer (which can bind up to 900 biotin molecules). This allows multiple SAPE molecules to bind to the biotinylated dendrimer through biotin-streptavidin interactions, thereby increasing the signal from the assay.

In some embodiments, the analytical method for detecting and quantifying the level of at least one target gene in the methods described herein is by gel electrophoresis and the use of labeled probes (e.g., probes labeled with radioactive or chemiluminescent tags), such as by Northern blot detection. In some embodiments, total RNA is isolated from the sample and then separated by size by SDS polyacrylamide gel electrophoresis. The separated RNA is then blotted on a membrane and hybridized with a radiolabeled complementary probe. In some embodiments, exemplary probes contain one or more affinity-enhanced nucleotide analogs as discussed below, such as locked nucleic acid ("LNA") analogs, which contain a bicyclic sugar moiety instead of deoxyribose or ribose. See, for example, Várallyay, E. et al. (2008) Nature Protocols 3(2): 190-196, which is incorporated herein by reference in its entirety.

In some embodiments, the detection and quantification of more than one target gene are achieved using microfluidic devices and single molecule detection. In some embodiments, the target RNA in the isolated total RNA sample is hybridized with two probes, one of which is complementary to the nucleic acid at the 5'-end of the target RNA and the second is complementary to the 3'-end of the target RNA. In some embodiments, each probe comprises more than one affinity-enhanced nucleotide analogs, such as LNA nucleotide analogs and each is labeled with a different fluorescent dye (i.e., a dye that is distinguished in detection) with a different fluorescence emission spectrum. The sample is then flowed through a microfluidic capillary, in which a variety of lasers excite the fluorescent probe, so that the unique coincidence burst of photons identifies the specific target RNA, and the unique coincidence burst number of specific photons can be counted to quantify the amount of target RNA in the sample. In some alternative embodiments, the target RNA-specific probe can be labeled with more than three different tags selected from, for example, fluorophores, electron spin labels, etc., and then hybridized with the RNA sample.

Optionally, the sample RNA is modified before hybridization. The target RNA/probe duplex is then passed through a channel in a microfluidic device containing detectors that record the unique signals of the three labels. In this way, individual molecules are detected and counted by their unique signals. See Fuchs et al., U.S. Genomics, Inc., U.S. Patent Nos. 7,402,422 and 7,351,538, each of which is incorporated herein by reference in its entirety.

4.2.5 Exemplary Automation and Systems

In some embodiments, gene expression is detected using automated sample manipulation and/or analytical platforms. In some embodiments, commercially available automated analytical platforms are utilized. For example, in some embodiments, a system (Cepheid, Sunnyvale, CA) is utilized.

The present invention is described using the GeneXpert system. Exemplary sample preparation and analysis methods are described below. However, the present invention is not limited to a particular detection method or analysis platform. Those skilled in the art will recognize that any number of platforms and methods may be utilized.

Utilizing a self-contained, single-use cartridge, sample extraction, amplification, and detection can all be performed within the self-contained "lab-in-a-cartridge" (see, e.g., U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which is incorporated herein by reference in its entirety).

The cartridge components include, but are not limited to, processing chambers containing reagents, filters, and capture technology for extracting, purifying, and amplifying target nucleic acids. Valves enable fluid transfer between chambers and contain nucleic acid cleavage and filtration components. Optical windows enable real-time optical detection. Reaction tubes enable very rapid thermal cycling.

In some embodiments, the system includes multiple modules for scalability. Each module includes multiple cartridges, along with sample handling and analysis components.

After the sample is added to the cartridge, it is contacted with a lysis buffer and the released DNA is bound to a DNA-binding matrix such as silica or a glass matrix. The sample supernatant is then removed and the DNA is eluted in an elution buffer such as Tris/EDTA buffer. The eluate can then be processed in the cartridge to detect the target gene as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents present in the cartridge as lyophilized particles.

In some embodiments, RT-PCR is used to amplify and analyze the presence of the target gene. In some embodiments, reverse transcription uses MMLV reverse transcriptase and is incubated at 40°C to 50°C for 5 to 20 minutes. In some embodiments, PCR uses Taq polymerase with hot start function, such as AptaTaq (Roche). In some embodiments, the initial denaturation is at 90°C to 100°C for 20 seconds to 5 minutes; the cyclic denaturation temperature is 90°C to 100°C for 1 to 10 seconds; the cyclic annealing and amplification temperature is 60°C to 75°C for 10 to 40 seconds; and up to 50 cycles are performed.

In some embodiments, a double denaturation method is used to amplify low copy number targets. In some embodiments, the double denaturation method includes a first denaturation step, followed by the addition of primers and/or probes for detecting the target gene. All or most of the DNA-containing samples (such as DNA eluates) are then denatured for a second time, after which, in some cases, a portion of the sample is aliquoted for circulation and detection of the target gene. Without wishing to be limited by any particular theory, the double denaturation scheme can increase the chance that the low copy number target gene (or its complement) will be present in the aliquot selected for circulation and detection because the second denaturation effectively doubles the number of targets (i.e., it divides the target and its complement into two templates) before selecting the aliquot for circulation. In some embodiments, the first denaturation step includes heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step includes heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first denaturation step and/or the second denaturation step are performed by heating aliquots of the sample separately. In some embodiments, each aliquot can be heated as listed above. As a non-limiting example, a first denaturation step for a DNA-containing sample (such as a DNA eluate) can include heating at least one, at least two, at least three, or at least four aliquots of the sample separately (sequentially or simultaneously) to a temperature of 90°C to 100°C for 60 seconds. As a non-limiting example, a second denaturation step for a DNA-containing sample (such as a DNA eluate) containing an enzyme, primer, and probe can include heating at least one, at least two, at least three, or at least four aliquots of the eluate separately (sequentially or simultaneously) to a temperature of 90°C to 100°C for 5 seconds. In some embodiments, the aliquot is the entire DNA-containing sample (such as a DNA eluate). In some embodiments, the aliquot is less than the entire DNA-containing sample (such as a DNA eluate).

In some embodiments, the following protocol is used to detect a target gene in a DNA-containing sample, such as a DNA eluate: one or more aliquots of the DNA-containing sample are each heated separately to 95°C for 60 seconds. An enzyme, primers, and probe are added to the DNA-containing sample and one or more aliquots are each heated separately to 95°C for 5 seconds. At least one aliquot of the DNA-containing sample containing the enzyme, primers, and probe is then heated to 94°C for 60 seconds. The aliquots are then cycled 45 times using the following 2-step cycle: (1) 94°C for 5 seconds, (2) 66°C for 30 seconds.

The present invention is not limited to specific primer and/or probe sequences. Exemplary amplification primers and detection probes are described in the Examples.

In some embodiments, off-line centrifugation is used, for example, to utilize samples with low cell content. The sample (with or without buffer) is centrifuged and the supernatant is removed. The pellet is then resuspended in a smaller volume of supernatant or buffer. The resuspended pellet is then analyzed as described herein.

4.2.6 Exemplary Data Analysis

In some embodiments, the presence of influenza is detected if the Ct value of any one of the influenza target genes (such as PA, PB2, MP, or NS) is below a certain threshold. In some embodiments, the effective range of Ct values is 12 to 39.9 Ct. In some such embodiments, if no amplification above background from influenza-specific primers is observed after 40 cycles, the sample is considered influenza-negative. In some such embodiments, if only the amplification of the exogenous control (SPC) exceeds background, the sample is considered influenza-negative.

In some embodiments, computer-based analysis program is used to translate the raw data produced by the detection assay into clinician's predicted value data. The clinician can use any appropriate means to obtain predicted data. Therefore, in some embodiments, the present invention provides further such benefits: the clinician (he may not have training in genetics or molecular biology) does not need to understand the raw data. Data are directly provided to the clinician in its most useful form. The clinician can then utilize this information immediately, thereby optimizing the care to the experimenter.

The present invention considers and can to and from the laboratory that measures, information provides, medical individual and experimenter receive, process and any method of transmitting information.For example, in some embodiments of the present invention, sample (for example, biopsy or serum or urine sample) is obtained from experimenter and offers analysis service provider (for example, clinical laboratory at medical facility, genome analysis business etc.) (being positioned at any part in the world (for example, being different from the country where experimenter is at or the country that information is finally used)) to produce raw data.In the case that sample comprises tissue or other biological sample, experimenter can visit medical center to obtain sample and be sent to analysis center, or experimenter can collect sample (for example, urine sample or sputum sample) and directly send it to analysis center.In the case that sample comprises the biological information determined before, can be directly sent to analysis service provider (for example, the information card containing information can be scanned by computer and use electronic communication system that data is transferred to the computer of analysis center) by experimenter.Once analysis center receives, sample is processed and produces specific analysis result (that is, expression data) for diagnosis required for experimenter or prognostic information.

The analysis data is then prepared in a form suitable for being interpreted by a treatment clinician. For example, rather than providing raw expression data, the prepared form can represent a diagnosis or risk assessment (for example, the presence of influenza) to the subject (with or without the advice selected for a specific treatment). The data can be presented by a clinician by any suitable method. For example, in some embodiments, an analysis service provider produces a report that can be printed for a clinician (for example, at a point of care) or displayed to the clinician on a computer monitor.

In some embodiments, first analyze information at the care point or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or the raw data is converted into information available to a clinician or patient. The central processing facility provides the privacy of data analysis (all data are stored in a central facility with a safety program consistent with specifications), the advantages of speed and consistency. The central processing facility can then control the fate of the data after the treatment subject. For example, using an electronic communication system, the central facility can provide data to a clinician, subject, or researcher.

In some embodiments, the subject can directly obtain data using an electronic communication system. The subject can be selected based on the results to further intervene or consult. In some embodiments, the data are used for research purposes. For example, the data can be used to further optimize the inclusion or exclusion of markers (as useful indications of a particular condition or disease stage or as companion diagnostics for a therapeutic process that determines the effect).

4.2.7 Exemplary Polynucleotides

In some embodiments, polynucleotides are provided. In some embodiments, synthetic polynucleotides are provided. As used herein, synthetic polynucleotides refer to polynucleotides synthesized chemically or enzymatically in vitro. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer such as OligoPilot (GE Healthcare), ABI 3900 DNA synthesizer (Applied Biosystems). Enzymatic synthesis includes, but is not limited to, amplification by enzymatic methods, for example, PCR, to produce polynucleotides. Polynucleotides may comprise one or more nucleotide analogs (i.e., modified nucleotides) discussed herein.

In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of an influenza A acidic polymerase (PA) gene. In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or complementary to, an influenza A acidic polymerase (PA) gene spanning 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides. Non-limiting exemplary polynucleotides are shown in Table A.

In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of an influenza A alkaline polymerase 2 (PB2) gene. In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to or complementary to, a region that is at least 90%, at least 95%, or 100% complementary to, a region that is 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of an influenza A alkaline polymerase 2 (PB2) gene. Non-limiting exemplary polynucleotides are shown in Table A.

In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of an influenza A1 matrix protein (MP) gene, an influenza A2 matrix protein (MP) gene, an influenza A3 hemagglutinin (HA) gene, an influenza B nonstructural protein (NS) gene, an RSV A genome, or an RSV B genome. In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical or complementary to an influenza A 1 matrix protein (MP) gene, an influenza A 2 matrix protein (MP) gene, an influenza A 3 hemagglutinin (HA) gene, an influenza B nonstructural protein (NS) gene, an RSV A genome, or an RSV B genome spanning 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides. Non-limiting exemplary polynucleotides are shown in Table B.

In various embodiments, the polynucleotide comprises fewer than 500, fewer than 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75, fewer than 50, fewer than 40, or fewer than 30 nucleotides. In various embodiments, the polynucleotide is 6 to 200, 8 to 200, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 40, 8 to 30, 15 to 100, 15 to 75, 15 to 50, 15 to 40, or 15 to 30 nucleotides in length.

In some embodiments, the polynucleotide is a primer. In some embodiments, the primer is labeled with a detectable portion. In some embodiments, the primer is unlabeled. Primer, as used herein, is a polynucleotide that can selectively hybridize with a target RNA or with a cDNA reverse transcribed from a target RNA or with an amplicon (collectively referred to as a "template") amplified from a target RNA or cDNA, and, in the presence of a template, a polymerase and appropriate buffer and reagents can extend from the primer extension product.

In some embodiments, polynucleotide is a probe. In some embodiments, the probe is labeled with a detectable moiety. Detectable moiety, as used herein, includes a direct detectable moiety, such as a fluorescent dye, and an indirect detectable moiety, such as a right member in conjunction with a pair. In some embodiments, when the detectable moiety is a right member in conjunction with a pair, the probe can be detected by incubating the probe with a detectable label that is incorporated into a second right member in conjunction with the probe. In some embodiments, the probe is unlabeled, such as when the probe is a capture probe, for example, on a microarray or a bead. In some embodiments, the probe is non-extendable (for example, by a polymerase). In other embodiments, the probe is extendable.

In some embodiments, the polynucleotide is a FRET probe that is labeled with a fluorescent dye (donor) at the 5' end and a quencher (acceptor) at the 3' end. The quencher is a chemical group that absorbs (i.e., suppresses) the fluorescence emitted from the dye when the group is in close proximity (i.e., attached to the same probe). Therefore, in some embodiments, the emission spectrum of the dye should overlap significantly with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the ends of the FRET probe.

4.2.7.1 Exemplary Polynucleotide Modifications

In some embodiments, the methods for detecting at least one target gene described herein utilize one or more modified polynucleotides, such as polynucleotides comprising one or more affinity-enhanced nucleotide analogs. Modified polynucleotides for use in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhanced nucleotides increases the binding affinity and specificity of the polynucleotide for its target nucleic acid compared to a polynucleotide containing only deoxyribonucleotides, and allows the use of shorter polynucleotides or shorter regions of complementarity between the polynucleotide and the target nucleic acid.

In some embodiments, affinity-enhanced nucleotide analogs include nucleotides comprising one or more base modifications, sugar modifications, and/or backbone modifications.

In some embodiments, modified bases for affinity-enhanced nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine.

In some embodiments, affinity-enhanced nucleotide analogs include nucleotides with modified sugars such as 2'-substituted sugars, such as 2'-O-alkyl-ribose sugars, 2'-amino-deoxyribose sugars, 2'-fluoro-deoxyribose sugars, 2'-fluoro-arabinose sugars, and 2'-O-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, the modified sugar is an arabinose sugar or a d-arabino-hexitol sugar.

In some embodiments, affinity-enhanced nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNA; e.g., oligomers comprising nucleobases linked together by an amino acid backbone. Other backbone modifications include phosphorothioate linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methyl phosphonates, alkyl phosphonates, phosphates, alkyl phosphonates, phosphoramides, carbamates, carbonates, phosphotriesters, acetamides, carboxymethyl esters, methyl phosphorothioates, phosphorodithioates, p-ethoxy groups, and combinations thereof.

In some embodiments, a polynucleotide includes at least one affinity-enhanced nucleotide analog with a modified base, at least one nucleotide with a modified sugar and/or at least one non-naturally occurring internucleotide linkage (which may be the same nucleotide).

In some embodiments, the affinity-enhanced nucleotide analogs contain locked nucleic acid ("LNA") sugars that are bicyclic sugars. In some embodiments, the polynucleotides used in the methods described herein comprise one or more nucleotides having LNA sugars. In some embodiments, the polynucleotides contain one or more regions consisting of nucleotides having LNA sugars. In other embodiments, the polynucleotides contain nucleotides having LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11): 1138-1142.

4.2.7.2 Exemplary Primers

In some embodiments, primers are provided. In some embodiments, primers are at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of influenza A acid polymerase (PA) gene at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary. In some embodiments, primers are provided that comprise a span of at least 90%, at least 95%, or 100% identical or complementary to 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of influenza A acid polymerase (PA) gene. Non-limiting exemplary primers are shown in Table A. In some embodiments, the primers may also include portions or regions that are not identical or complementary to the target gene. In some embodiments, regions of the primers that are at least 90%, at least 95%, or 100% identical or complementary to the target gene are adjacent, such that any regions of the primers that are not identical or complementary to the target gene do not disrupt the regions of identity or complementarity.

In some embodiments, primers are provided. In some embodiments, the primers are at least 90%, at least 95%, or 100% identical to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the influenza A alkaline polymerase 2 (PB2) gene, or at least 90%, at least 95%, or 100% complementary. In some embodiments, primers are provided that comprise a region that is at least 90%, at least 95%, or 100% identical or complementary to a span of 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of the influenza A alkaline polymerase 2 (PB2) gene. Non-limiting exemplary primers are shown in Table A. In some embodiments, the primers may also include portions or regions that are not identical or complementary to the target gene. In some embodiments, the regions of the primers that are at least 90%, at least 95%, or 100% identical or complementary to the target gene are adjacent, such that the regions of the primers that are not identical or complementary to the target gene do not disrupt the identical or complementary regions.

In some embodiments, primers are provided. In some embodiments, the primers are at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of an influenza A1 matrix protein (MP) gene, an influenza A2 matrix protein (MP) gene, an influenza A3 hemagglutinin (HA) gene, an influenza B nonstructural protein (NS) gene, an RSV A genome, or an RSV B genome. In some embodiments, primers are provided that comprise a span of at least 90%, at least 95%, or 100% identical or complementary regions to influenza A 1 matrix protein (MP) gene, influenza A 2 matrix protein (MP) gene, influenza A 3 hemagglutinin (HA) gene, influenza B nonstructural protein (NS) gene, RSV A genome, or RSV B genome of 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides. Non-limiting exemplary primers are shown in Table B. In some embodiments, primers can also comprise a portion or region that is not identical or non-complementary to the target gene. In some embodiments, the region of primers that is at least 90%, at least 95%, or 100% identical or complementary to the target gene is adjacent, such that the region of primers that is not identical or non-complementary to the target gene does not destroy the identical or complementary region.

In some embodiments, the primer comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene. In some such embodiments, the primer comprising a region that is at least 90%, at least 95%, or 100% identical to a region of the target gene is capable of selectively hybridizing to a cDNA reverse transcribed from RNA, or to an amplicon produced by amplification of the target gene. In some embodiments, the primer is complementary to a sufficient portion of the cDNA or amplicon so that it selectively hybridizes to the cDNA or amplicon under the specific assay conditions used.

As used herein, "selectively hybridizes" means that a polynucleotide, such as a primer or probe, will hybridize to a specific nucleic acid in a sample with at least 5-fold greater affinity than it will hybridize to another nucleic acid present in the same sample that has a different nucleotide sequence in the hybridization region. Exemplary hybridization conditions are discussed herein, for example, in the context of a reverse transcription reaction or a PCR amplification reaction. In some embodiments, a polynucleotide will hybridize to a specific nucleic acid in a sample with at least 10-fold greater affinity than it will hybridize to another nucleic acid present in the same sample that has a different nucleotide sequence in the hybridization region.

In some embodiments, primers are used to reverse transcribe a target RNA, e.g., as discussed herein. In some embodiments, primers are used to amplify a target RNA or a cDNA reverse transcribed therefrom. In some embodiments, the amplification is quantitative PCR, e.g., as discussed herein.

In some embodiments, the primer comprises a detectable moiety.

In some embodiments, primer pairs are provided. The primer pairs are designed to amplify a target gene, such as the influenza A PA gene, the influenza A PB2 gene, the influenza A 1 matrix protein (MP) gene, the influenza A 2 matrix protein (MP) gene, the influenza A 3 hemagglutinin (HA) gene, the influenza B nonstructural protein (NS) gene, the RSV A genome, or the RSV B genome, or a portion of an endogenous control (such as a sample adequacy control (SAC)), or an exogenous control (such as a sample processing control (SPC)). In some embodiments, primers are designed to produce an amplicon that is 50 to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150 nucleotides long. Non-limiting exemplary primers are shown in Tables A and B.

4.2.7.3 Exemplary Probes

In various embodiments, the method for detecting the presence of influenza, and optionally, RSV, comprises hybridizing the nucleic acid of the sample with a probe. In some embodiments, the probe comprises a portion complementary to a target gene, such as the influenza A PA gene or the influenza A PB2 gene, or an endogenous control such as a sample adequacy control (SAC), or an exogenous control such as a sample processing control (SPC). In some embodiments, the probe comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene. In some such embodiments, a probe that is at least 90%, at least 95%, or 100% complementary to a target gene is complementary to a sufficient portion of the target gene so that it selectively hybridizes to the target gene under the specific assay conditions used. In some embodiments, a probe that is complementary to a target gene comprises a region that is at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the target gene. Non-limiting exemplary probes are shown in Tables A and B. A probe that is at least 90%, at least 95%, or 100% complementary to a target gene may also comprise a portion or region that is not complementary to the target gene. In some embodiments, the region of the probe that is at least 90%, at least 95%, or 100% complementary to the target gene is contiguous, such that any region of the probe that is not complementary to the target gene does not disrupt the complementary region.

In some embodiments, the probe comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene, or an endogenous control such as a sample adequacy control (SAC), or an exogenous control such as a sample processing control (SPC). In some such embodiments, the probe comprising a region that is at least 90%, at least 95%, or 100% identical to a region of the target gene is capable of selectively hybridizing to a cDNA reverse transcribed from the target gene or to an amplicon produced by amplifying the target gene. In some embodiments, the probe is at least 90%, at least 95%, or 100% complementary to a sufficient portion of the cDNA or amplicon such that it selectively hybridizes to the cDNA or amplicon under the specific assay conditions being used. In some embodiments, the probe that is complementary to the cDNA or amplicon comprises a region that is at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the cDNA or amplicon. The probe that is at least 90%, at least 95%, or 100% complementary to the cDNA or amplicon can also comprise a portion or region that is not complementary to the cDNA or amplicon. In some embodiments, the region of the probe that is at least 90%, at least 95%, or 100% complementary to the cDNA or amplicon is adjacent such that any region of the probe that is not complementary to the cDNA or amplicon does not disrupt the region of complementarity.

In some embodiments, the method of detecting more than one target gene comprises: (a) reverse transcribing the target RNA to produce cDNA complementary to the target RNA; (b) amplifying the cDNA from (a); and (c) detecting the amount of the target RNA using real-time RT-PCR and a detection probe (which can be simultaneous with the amplification step (b)).

As described above, in some embodiments, real-time RT-PCR detection can be carried out using FRET probes, which include, but are not limited to, probes, molecular beacon probes and Scorpion probes. In some embodiments, real-time RT-PCR detection is carried out using probes, which typically have a fluorescent dye covalently bound at one end of DNA and a linear probe covalently bound to a quencher molecule at other locations (such as at the other end of DNA). FRET probes include sequences complementary to the region of cDNA or amplicon, so that when FRET probes hybridize with cDNA or amplicon, dye fluorescence is quenched, and when probes are digested during the amplification of cDNA or amplicon, dye is released from probe and produces fluorescent signal. In some embodiments, the amount of target gene in sample is proportional to the amount of fluorescence measured during amplification.

The probe typically comprises a region of contiguous nucleotides having a sequence that is at least 90%, at least 95%, or 100% identical or complementary to a region of the target gene or its complementary cDNA reverse transcribed from a target RNA template (i.e., the sequence of the probe region is complementary to or identical to the target RNA to be detected and present in the target RNA to be detected), such that the probe can selectively hybridize with a PCR amplicon of a region of the target gene. In some embodiments, the probe comprises a region of at least 6 contiguous nucleotides having a sequence that is completely complementary to or identical to a region of the cDNA reverse transcribed from the target gene and present in the region of the cDNA reverse transcribed from the target gene. In some embodiments, the probe comprises a region of at least 8 contiguous nucleotides, at least 10 contiguous nucleotides, at least 12 contiguous nucleotides, at least 14 contiguous nucleotides, or at least 16 contiguous nucleotides that are at least 90%, at least 95%, or 100% identical or complementary to a region of the cDNA reverse transcribed from the target gene and present in the region of the cDNA reverse transcribed from the target gene and present.

In some embodiments, the region of the amplicon having a sequence that is at least 90%, at least 95%, or 100% complementary to the probe sequence is at or near the center of the amplicon molecule. In some embodiments, at least two nucleotides, such as at least three nucleotides, such as at least four nucleotides, such as at least five nucleotides, of the amplicon are independently present at the 5'-end and the 3'-end of the region of complementarity.

In some embodiments, molecular beacons can be used to detect PCR products. Similar to probes, molecular beacons use FRET to detect PCR products via probes with fluorescent dyes and quenchers attached to the ends of the probes. Unlike probes, molecular beacons remain intact during PCR cycles. When free in solution, molecular beacon probes form a stem-loop structure, allowing the dye and quencher to be close enough to cause fluorescence quenching. When the molecular beacon hybridizes to the target, the stem-loop structure is eliminated, thereby separating the dye and quencher in space, and the dye emits fluorescence. Molecular beacons can be obtained from, for example, Gene Link ^™ (see www.genelink.com/newsite/products/mbintro.asp).

In some embodiments, Scorpion probes can be used as sequence-specific primers and for PCR product detection. Like molecular beacons, when not hybridizing with target nucleic acid, Scorpion probes form a stem-loop structure. However, unlike molecular beacons, Scorpion probes achieve both sequence-specific initiation and PCR product detection. A fluorescent dye molecule is attached to the 5'-end of the Scorpion probe, and a quencher is attached elsewhere, such as the 3'-end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and the complementary portion is connected to the 5'-end of the probe by a non-amplifiable portion. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement in the extended amplicon, thereby opening the stem-loop structure and allowing the dye at the 5'-end to fluoresce and generate a signal. The Scorpion probe can be obtained from, for example, Premier Biosoft International (see www.premierbiosoft.com/tech_notes/Scorpion.html).

In some embodiments, labels that can be used on FRET probes include colorimetric and fluorescent dyes such as AlexaFluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarins and their derivatives, such as 7-amino-4-methylcoumarin, aminocoumarins, and hydroxycoumarins; cyanine dyes, such as Cy3 and Cy5; eosin and true eosin; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye ^™ ; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine, and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, those described above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2',4',5',7'-tetrabromosulfone fluorescein, and TET.

Examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, but are not limited to, fluorescein/tetramethylrhodamine; IAEDANS/fluorescein; EDANS/dabcyl; fluorescein/fluorescein; BODIPY FL/BODIPY FL; fluorescein/QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, FRET can be detected by fluorescence depolarization in some embodiments. Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor 568/Alexa Fluor 647; Alexa Fluor 594/Alexa Fluor 647; Alexa Fluor 350/Alexa Fluor 488; Alexa Fluor 488/Alexa Fluor 546; Alexa Fluor 488/Alexa Fluor 555; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor 488/Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647; Alexa Fluor 546/Alexa Fluor 568; Alexa Fluor 546/Alexa Fluor 594; Alexa Fluor 546/Alexa Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor 555/Alexa Fluor 647; Alexa Fluor 568/Alexa Fluor 647; Alexa Fluor 594/Alexa Fluor 647; Alexa Fluor 350/Alexa Fluor 488; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor 568/QSY 7 or QSY 9; Alexa Fluor 568/QSY 21; Alexa Fluor 594/QSY 21; and Alexa Fluor 647/QSY 21. In some cases, the same quencher can be used for multiple dyes, for example, a spectral quencher such as Iowa Quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole Quencher ^™ (BHQ ^™ ; Sigma-Aldrich, St. Louis, MO).

In some embodiments, for example, in a multiplex reaction in which two or more moieties (e.g., amplicons) are detected simultaneously, each probe comprises a detectably distinct dye, such that the dyes can be distinguished when detected simultaneously in the same reaction. One skilled in the art can select a panel of detectably distinct dyes for use in a multiplex reaction.

Specific examples of fluorescently labeled ribonucleotides that can be used to prepare PCR probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include Alexa Fluor 488-5-UTP, fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides for use in preparing PCR probes for use in the methods described herein include dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, rhodamine green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, for example, Invitrogen.

In some embodiments, dye and other parts, such as quencher, are introduced into the polynucleotide for methods described herein, such as FRET probe by the nucleotide of modification." modified nucleotide " refers to chemically modified, but still works as nucleotide nucleotide. In some embodiments, the nucleotide of modification has a covalently linked chemical part such as dye or quencher, and can, for example, be introduced into the polynucleotide by the mode of solid phase synthesis polynucleotide. In other embodiments, the nucleotide of modification includes more than one reactive group reacting with dye or quencher before, during or after the nucleotide of modification is incorporated into nucleic acid. In a specific embodiment, the nucleotide of modification is an amine-modified nucleotide, that is, modified to have a nucleotide with reactive amine groups. In some embodiments, the nucleotide of modification includes a base moiety modified, such as uridine, adenosine, guanosine, and/or cytosine. In specific embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxo-bis-(ethylamine)]-CTP; N6-(6-amino)hexyl-ATP; 8-[(6-amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP is substituted for 5-(3-aminoallyl)-UTP. Many amine-modified nucleotides are commercially available from, for example, Applied Biosystems, Sigma, Jena Bioscience, and TriLink.

Exemplary detectable moieties also include, but are not limited to, members of a binding pair. In some such embodiments, the first member of the binding pair is attached to a polynucleotide. The second member of the binding pair is attached to a detectable label, such as a fluorescent label. When the polynucleotide attached to the first member of the binding pair is incubated with the second member of the binding pair attached to the detectable label, the first and second members of the binding pair associate and the polynucleotide can be detected. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, and the like.

In some embodiments, multiple target genes are detected in a single multiplex reaction. In some such embodiments, each probe targeting a unique amplicon is spectrally distinguishable when released from the probe, in which case each target gene is detected by a unique fluorescent signal. In some embodiments, the same fluorescent signal is used to detect two or more target genes, in which case detection of the signal indicates the presence of one or both target genes.

Those skilled in the art can select an appropriate detection method for the selected assay, for example, a real-time RT-PCR assay.The selected detection method need not be the method described above, and can be any method.

4.3 Exemplary Compositions and Kits

In another aspect, compositions are provided. In some embodiments, compositions are provided for use in the methods described herein.

In some embodiments, a composition is provided, which comprises at least one target gene-specific primer. The terms "target gene-specific primer" and "target RNA-specific primer" can be used interchangeably and include primers for a continuous nucleotide region with the following sequences: (i) at least 90%, at least 95%, or 100% identical to the region of the target gene, or (ii) a sequence at least 90%, at least 95%, or 100% complementary to the sequence of the continuous nucleotide region present in the target gene. In some embodiments, a composition is provided, which comprises at least one target gene-specific primer pair. The term "target gene-specific primer pair" includes primer pairs suitable for amplifying a defined target gene region. A target gene-specific primer pair typically includes: a first primer comprising a sequence at least 90%, at least 95%, or 100% identical to the sequence of the region of the target gene and a second primer comprising a sequence at least 90%, at least 95%, or 100% complementary to the region of the target gene. Primer pairs are generally suitable for amplifying a region of a target gene that is 50 to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150 nucleotides long. Non-limiting exemplary primers are shown in Tables A and B for primer pairs.

In some embodiments, the composition comprises at least one target gene-specific primer pair. In some embodiments, the composition further comprises a target gene-specific primer pair for amplifying an endogenous control (such as SAC) and/or a pair of target gene-specific primers for amplifying an exogenous control (such as SPC).

In some embodiments, the composition comprises at least one target gene-specific probe. The terms "target gene-specific probe" and "target RNA-specific probe" are used interchangeably and include probes having a contiguous nucleotide region of the following sequence: (i) at least 90%, at least 95%, or 100% identical to a region of the target gene, or (ii) at least 90%, at least 95%, or 100% complementary to the sequence of a contiguous nucleotide region present in the target gene. Non-limiting exemplary target-specific probes are shown in Tables A and B.

In some embodiments, the composition (including the compositions described above comprising one or more target gene-specific primer pairs) comprises one or more probes for detecting the target gene. In some embodiments, the composition comprises a probe for detecting an endogenous control (such as SAC) and/or a probe for detecting an exogenous control (such as SPC).

In some embodiments, the composition is an aqueous composition. In some embodiments, the aqueous composition comprises a buffer component, such as phosphate, tris, HEPES, etc., and/or additional components, as discussed below. In some embodiments, the composition is dry, for example, lyophilized, and suitable for reconstitution by adding a liquid. The dry composition can include one or more buffer components and/or additional components.

In some embodiments, the composition further comprises one or more additional components. Such additional components include, but are not limited to, salts such as NaCl, KCl, and _MgCl2 ; polymerases, including thermostable polymerases such as Taq; dNTPs; reverse transcriptases such as MMLV reverse transcriptase; RNase inhibitors; bovine serum albumin (BSA); reducing agents such as β-mercaptoethanol; EDTA; and the like. Those skilled in the art can select appropriate composition components based on the intended use of the composition.

In some embodiments, a composition is provided, which comprises at least one polynucleotide for detecting at least one target gene. In some embodiments, the polynucleotide is used as a primer for a reverse transcriptase reaction. In some embodiments, the polynucleotide is used as a primer for amplification. In some embodiments, the polynucleotide is used as a primer for PCR. In some embodiments, the polynucleotide is used as a probe for detecting at least one target gene. In some embodiments, the polynucleotide is detectably labeled. In some embodiments, the polynucleotide is a FRET probe. In some embodiments, the polynucleotide is a probe, a molecular beacon, or a Scorpion probe.

In some embodiments, the composition comprises at least one FRET probe having a sequence that is at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to a region of a target gene (such as the influenza A PA gene or the influenza A PB2 gene). In some embodiments, the FRET probe is labeled with a donor/acceptor pair such that when the probe is digested during the PCR reaction, it produces a unique fluorescence emission associated with the specific target gene. In some embodiments, when the composition comprises multiple FRET probes, each probe is labeled with a different donor/acceptor pair such that when the probe is digested during the PCR reaction, each produces a unique fluorescence emission associated with a specific probe sequence and/or target gene. In some embodiments, the sequence of the FRET probe is complementary to the target region of the target gene. In other embodiments, the FRET probe has a sequence that contains one or more base mismatches when compared to the sequence of the optimally aligned target region of the target gene.

In some embodiments, the composition comprises a FRET probe consisting of at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides, wherein at least a portion of the sequence is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, a region of a target gene, such as an influenza A PA gene or an influenza A PB2 gene. In some embodiments, at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of the FRET probe are identically present in, or complementary to, a region of a target gene (such as an influenza A PA gene or an influenza A PB2 gene). In some embodiments, the FRET probe has a sequence that contains one, two, or three base mismatches when compared to the sequence or complement of the target gene.

In some embodiments, the test kit comprises the polynucleotides discussed above. In some embodiments, the test kit comprises at least one primer and/or probe discussed above. In some embodiments, the test kit comprises at least one polymerase, such as a thermostable polymerase. In some embodiments, the test kit comprises dNTPs. In some embodiments, the test kit for the real-time RT-PCR method described herein comprises one or more target gene-specific FRET probes and/or one or more primers for reverse transcription of target RNA and/or one or more primers for amplifying target genes or cDNA reversely transcribed therefrom.

In some embodiments, more than one primer and/or probe is "linear". A "linear" primer refers to a polynucleotide of a single-stranded molecule and generally does not include, for example, a short region of at least 3, 4 or 5 consecutive nucleotides (which is complementary to another region within the same polynucleotide so that the primer forms an internal duplex). In some embodiments, a primer for reverse transcription comprises a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more consecutive nucleotides at the 3'-end, which has a sequence complementary to a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more consecutive nucleotides at the 5'-end of the target gene.

In some embodiments, the reagents comprise one or more linear primer pairs ("forward primers" and "reverse primers") for amplifying a target gene or a cDNA reverse transcribed therefrom. Thus, in some embodiments, the first primer comprises a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides having a sequence that is at least 90%, at least 95%, or 100% identical to the sequence of a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides at a first position of the target gene. Furthermore, in some embodiments, the second primer comprises a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides having a sequence that is at least 90%, at least 95%, or 100% complementary to the sequence of a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides at a second position of the target gene, such that a PCR reaction using the two primers results in an amplicon extending from the first position of the target gene to the second position of the target gene.

In some embodiments, the kit comprises at least two, at least three, or at least four sets of primers, each set being used to amplify a different target gene or cDNA reverse transcribed therefrom. In some embodiments, the kit further comprises at least one set of primers for amplifying control RNA, such as an endogenous control and/or an exogenous control.

In some embodiments, probes and/or primers used in the compositions described herein comprise deoxyribonucleotides. In some embodiments, probes and/or primers used in the compositions described herein comprise deoxyribonucleotides and one or more nucleotide analogs, such as the LNA analogs or other duplex-stabilizing nucleotide analogs described above. In some embodiments, probes and/or primers used in the compositions described herein comprise all nucleotide analogs. In some embodiments, probes and/or primers comprise one or more duplex-stabilizing nucleotide analogs, such as LNA analogs, in the region of complementarity.

In some embodiments, the kit of the real-time RT-PCR method described herein further comprises reagents for reverse transcription and amplification reaction. In some embodiments, the kit comprises an enzyme, such as a reverse transcriptase or a thermostable DNA polymerase, such as Taq polymerase. In some embodiments, the kit further comprises deoxyribonucleotide triphosphates (dNTPs) for reverse transcription and/or amplification. In other embodiments, the kit comprises a buffer optimized for the specific hybridization of probes and primers.

A kit generally includes packaging having one or more containers for holding reagents, as one or more separate compositions or, optionally, as a mixture in which compatibility of the reagents is permitted. The kit may also include one or more other materials that may be desired from the user's perspective, such as one or more buffers, one or more diluents, one or more standards, and/or any other materials for sample processing, washing, or any other steps in performing the assay.

The kit preferably includes instructions for performing one or more of the methods described herein. The instructions included in the kit may be attached to the packaging material or may be included therein as a package insert. While the instructions are typically handwritten or printed material, they are not limited thereto. The present invention contemplates any medium capable of storing the instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, magnetic cylinders, magnetic chips), optical media (e.g., CD ROMs), etc. As used herein, the term "instructions" may include a website address where the instructions are provided.

In some embodiments, the test kit may include the reagents described above provided in one or more cartridges. These cartridges allow extraction, amplification, and detection to be performed within the self-contained "laboratory in a cartridge" (see, e.g., U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which is incorporated herein by reference in its entirety). Reagents for measuring genome copy number levels and detecting pathogens can be provided in separate cartridges within the test kit or can be provided in a single cartridge (suitable for multiplex detection).

In some embodiments, any kit described herein can include a container for a nasal aspirate/wash sample and/or a swab for collecting a nasopharyngeal swab sample.

The following examples are for illustrative purposes only and are not intended to be limiting in any way.

5. Examples

5.1 Example 1: Detection of Influenza A (FluA)

Many existing influenza tests rely on detection of the influenza matrix protein (MP) gene. The constant antigenic drift and shift of influenza viruses makes it difficult to maintain assay sensitivity between seasons, leading to deterioration in assay performance over time. To design robust influenza assays that are less likely to lose sensitivity, or lose sensitivity at a slower rate than existing tests, additional influenza A targets that could complement the MP have been investigated.

First, by using European Molecular Biology Laboratory (EMBL)-European Bioinformatics Institute (European Bioinformatics Institute) (EBI) sequence alignment software, ClustalW produces the sequence alignment of RNA segment, identifies the appropriate gene fragment for designing primers and probes.ClustalW is the general purpose multiple sequence alignment program for nucleic acid or protein, and it calculates the best match for the sequence selected, and they are compared, so that identity, similarity and difference can be compared.For each potential target, the sequence region (length 100-200nt) that selects to distinguish target.Also based on the frequency selection region of polymorphic base substitution; Select highly conservative region.

DNA Software, Inc.'s Visual OMP (Oligonucleotide Modeling Platform) is used to design primers and probes for amplifying RNA fragments in selected regions. Visual OMP models the folding and hybridization of single-stranded nucleic acids in silico by combining all public domain thermodynamic parameters as well as proprietary neighbor and polymorphic thermodynamic parameters for DNA, RNA, PNA, and inosine. This enables efficient design of primers and probes for complex assays such as microarrays, microfluidic applications, and multiplex PCR. Computer experiments simulate secondary structures given specific conditions for targets (optimal and suboptimal), primers (optimal and suboptimal), homodimers, and target and primer heterodimers. Values for melting temperature (Tm), free energy (ΔG), percent binding, and concentration are calculated for all species. In addition, Visual OMP predicts the binding efficiency between primers and probes and one or more targets in single or multiplex reactions.

Using this software tool, predicted interactions between oligonucleotides and different influenza targets were thermodynamically evaluated and undesired interactions were minimized.

The selected primers and probes were then subjected to BLAST searches, both individually and in combination, querying for oligonucleotides representing the expected full-length amplicon sequence.

Based on this analysis, two new influenza A target genes were identified that could improve the sensitivity of existing influenza assays: the basic polymerase 2 (PB2) gene and the acidic polymerase (PA) gene. Exemplary PB2 and PA gene sequences are shown in SEQ ID NOs: 1 and 2, respectively. Primers and probes for detecting the two new influenza A target genes were designed as described above and are shown in Table A.

Table A: Primers and probes for detecting influenza A 1PB2 and PA genes

In addition, primers and probes were designed as described above to detect influenza A1 matrix protein (MP) gene, influenza A2 (avian isolate) MP gene, influenza A3 (H7N9) hemagglutinin (HA) gene, influenza B MP gene, influenza B NS gene, and respiratory syncytial virus (RSV) A and B. Exemplary influenza A1 MP, influenza A2 MP, influenza A3 HA, influenza B MP, and influenza B NS gene sequences are shown in SEQ ID NOs: 3 to 7, respectively. Those primers and probes are shown in Table B.

Table B: For detection of influenza A 1MP, influenza A 2MP, influenza A 3HA, influenza B MP, influenza B Primers and probes for NS, RSV A, and RSV B

F1, F2, F3, F4 and F5 are detectably distinct dyes that can be simultaneously detected and distinguished in multiplex reactions. Each probe also contains a quencher (eg, Q1 or Q2, as described above).

The final primer and probe compositions for the multiplex assay are shown in Table B.

Table B: Primer and probe concentrations

Each reaction contained 50-90 mM KCl, 3-5 mM MgCl ₂ , 400-825 μM dNTPs, 20 mM Tris, pH 8.5, 0.01% sodium azide, and 1 unit/μl of RNase inhibitor. MMLV reverse transcriptase (2 units/μl) and AptaTaq (3 units/μl; Roche) were used for reverse transcription and amplification, respectively.

For NP swabs, place the swab sample in a tube containing 3 mL of transport medium. For NA/W samples, add 600 μL of NA/W sample to 3 mL of transport medium.

300 μL of buffered nasopharyngeal swab (NP) or nasal aspirate/wash sample (NA/W) samples were loaded into the cartridge for analysis. The sample was mixed with a lysis reagent to release nucleic acids. After lysis, the nucleic acids released from the sample were captured on a DNA-binding substrate. The nucleic acids were eluted from the substrate and used to reconstitute the reagents for real-time PCR (described above). The reaction cycle used was: 94°C for 20 seconds using the cartridge, followed by up to 3 cycles of 95°C for 1 second, 62°C for 35 seconds, up to 20 cycles of 95°C for 1 second, 62°C for 20 seconds, and 14 cycles of 95°C for 1 second, 62°C for 35 seconds.

The valid range of Ct values for the target was 12-39.9 Ct.

The Xpert Flu/RSV XC assay has three channels (Influenza A 1, Influenza A 2, and Influenza A 3) to detect the most influenza A strains. The primers and probes in the Influenza A 1 channel have 100% homology to human influenza A strains. The primers and probes in the Influenza A 2 channel have> 95% homology to avian influenza A strains and approximately 80% homology to human influenza A strains. The primers and probes in the Influenza A 3 channel detect the hemagglutinin gene segment (subtyping capability) of the avian influenza A H7N9 strain. All influenza A strains (human and avian) detected by the Xpert Flu/RSV XC assay are reported as influenza A positive.

In the Xpert Flu/RSV XC assay, the influenza A result retrieval algorithm requires either the influenza A 1 or influenza A 2 channel to be positive in order to report a positive influenza A test result. A positive influenza A 3 channel, with a positive influenza A 1 or influenza A 2 result, will not be reported as positive and will be reported as invalid. Table 1 below lists all possible test results for influenza A.

Table 1. Possible test results for influenza A for the influenza A 1, influenza A 2, and influenza A 3 channels.

All possible outcomes are shown in Tables 2 and 3.

Table 2. All possible final test results for the Xpert Flu-RSV XC Select assay

Table 3. Xpert Flu-RSV XC Assay Results and Interpretation

5.2 Example 2: Clinical manifestations

The performance characteristics of the Xpert Flu/RSV XC assay were evaluated at six institutions in the U.S. Due to the low prevalence of influenza viruses and the difficulty in obtaining fresh influenza and RSV-positive specimens, the sample population for this study was supplemented with cryopreserved specimens.

Subjects included individuals with signs and symptoms of respiratory infection who required routine care to collect nasal aspirate/wash (NA/W) samples or nasopharyngeal (NP) swab samples for influenza and RSV testing. For eligible subjects, an aliquot of the remaining sample was obtained for testing and reference testing using the Xpert Flu/RSV XC assay, and patient management continued at the site according to their standard practice.

The Xpert Flu/RSV XC assay performance was compared to an FDA-cleared comparator assay. In cases of discrepancies between the Xpert Flu/RSV XC assay and the comparator assay, bidirectional sequencing was performed on samples.

5.2.1 Nasal Aspirate/Wash (NA/W) Samples

A total of 657 NA/W samples were tested for influenza A, influenza B, and RSV by the Xpert Flu/RSV XC assay and the reference assay. Of the 657 NA/W samples, 581 were fresh, prospectively collected, and 76 were frozen-stored samples.

Overall, using NA/W samples, the Xpert Flu/RSV XC assay demonstrated 98.6%, 100%, and 99.8% positive agreement rates (PPA), negative agreement rates (NPA), and overall agreement rates (OPA) for the detection of influenza A, respectively, relative to the reference assay (Table 4). The PPA, NPA, and OPA of the Xpert Flu/RSV XC assay for influenza B were 99.2%, 100%, and 99.8%, respectively (Table 4). The PPA, NPA, and OPA of the Xpert Flu/RSV XC assay for RSV were 97.2%, 99.6%, and 99.1%, respectively (Table 4).

For fresh, prospectively collected NA/W samples, the Xpert Flu/RSV XC assay demonstrated 100%, 100%, and 100% PPA, NPA, and OPA, respectively, for detection of influenza A relative to the reference assay (Table 4). The Xpert Flu/RSVXC assay demonstrated 99.2%, 100%, and 99.8% PPA, NPA, and OPA, respectively, for influenza B (Table 4). The Xpert Flu/RSVXC assay demonstrated 98.5%, 99.6%, and 99.3% PPA, NPA, and OPA, respectively, for RSV (Table 4).

For frozen stored NA/W samples, the Xpert Flu/RSV XC assay demonstrated 97.1%, 100%, and 98.7% PPA, NPA, and OPA, respectively, for detection of influenza A relative to the reference assay (Table 4). The Xpert Flu/RSV XC assay demonstrated 100%, 100%, and 100% PPA, NPA, and OPA, respectively, for influenza B (Table 4). The Xpert Flu/RSV XC assay demonstrated 84.6%, 100%, and 97.4% PPA, NPA, and OPA, respectively, for RSV (Table 4).

Table 4. Xpert Flu/RSV XC Assay Performance for NA/W Samples

a. Results of sequencing tests: NA: The sample was not sequenced.

b. Results of sequencing tests: 2 out of 2 were RSV positive.

c. Results of sequencing tests: 1 out of 2 was RSV positive; 1 out of 2 was RSV negative.

d. Test results by sequencing: 1 out of 1 were negative for influenza A.

e. Results of sequencing tests: 1 out of 2 was RSV positive; 1 out of 2 was RSV negative.

f. Results of sequencing tests: 1 out of 1 were negative for influenza A.

g. Results of sequencing tests: NA: The sample was not sequenced.

h. Results of sequencing tests: 2 out of 2 were RSV positive.

i. Results of sequencing tests: 2 out of 4 were RSV positive; 2 out of 4 were RSV negative.

5.2.2 Nasal Aspirate/Wash (NA/W) Samples

A total of 593 NP swab specimens were tested for influenza A, influenza B, and RSV by the Xpert Flu/RSV XC assay and the reference assay. Of the 593 NP swab specimens, 190 were fresh, prospectively collected and 403 were frozen, stored specimens.

Overall, using NP swab specimens, the Xpert Flu/RSV XC assay demonstrated 98.1%, 95.1%, and 95.6% PPA, NPA, and OPA, respectively, for detection of influenza A compared to the reference assay (Table 5). The Xpert Flu/RSV XC assay demonstrated 98.9%, 100%, and 99.8% PPA, NPA, and OPA, respectively, for influenza B (Table 5). The Xpert Flu/RSV XC assay demonstrated 91.9%, 99.4%, and 98.7% PPA, NPA, and OPA, respectively, for RSV (Table 5).

For fresh, prospectively collected NP swab specimens, the Xpert Flu/RSV XC assay demonstrated 85.7%, 98.9%, and 98.4% PPA, NPA, and OPA, respectively, for detection of influenza A relative to the reference assay (Table 5). The Xpert Flu/RSV XC assay demonstrated 100%, 100%, and 100% PPA, NPA, and OPA, respectively, for influenza B (Table 5). The Xpert Flu/RSV XC assay demonstrated 100%, 100%, and 100% PPA, NPA, and OPA, respectively, for RSV (Table 5).

For frozen, stored NP swab specimens, the Xpert Flu/RSV XC assay demonstrated 99.0%, 92.8%, and 94.3% PPA, NPA, and OPA, respectively, for detection of influenza A relative to the reference assay (Table 5). The Xpert Flu/RSVXC assay demonstrated 98.8%, 100%, and 99.8% PPA, NPA, and OPA, respectively, for influenza B (Table 5). The Xpert Flu/RSVXC assay demonstrated 90.4%, 99.1%, and 98.0% PPA, NPA, and OPA, respectively, for RSV (Table 5).

Table 5. Xpert Flu/RSV XC Assay Performance on NP Swab Specimens

a. Results of sequencing tests: 2 out of 2 were positive for influenza A (Flu A).

b. Sequencing test results: 1 out of 1 tested negative for influenza A.

c. Results of sequencing tests: 17 out of 22 were positive for influenza A; 5 out of 22 were negative for influenza A.

d. Test results by sequencing: 1 out of 1 were negative for influenza A.

e. Results of sequencing tests: 1 out of 1 were negative for influenza B (Flu B).

f. Results of sequencing tests: 2 out of 3 were RSV positive; 1 out of 3 was RSV negative.

g. Results of sequencing tests: 1 out of 5 was RSV positive; 4 out of 5 were RSV negative.

h. Results of sequencing tests: 19 of 24 were positive for influenza A; 5 of 24 were negative for RSV.

i. Results of sequencing tests: 2 out of 2 were negative for influenza A.

j. Test results by sequencing: 1 out of 1 were negative for influenza B.

k. Results of sequencing tests: 2 out of 3 were RSV positive; 1 out of 3 was RSV negative.

l. Results of sequencing tests: 1 out of 5 was RSV positive; 4 out of 5 were RSV negative.

In the Xpert Flu/RSV XC assay run using qualified samples, 98.6% (1236/1254) of these samples were successful on the first attempt. The remaining 18 gave inconclusive results on the first attempt (11 errors, 3 invalid results, and 4 no results). Seventeen of the 18 samples were retested, and 14 of them gave valid results after a single retest. There were four inconclusive NA/W samples after retesting, and they were excluded from the analysis.

5.3. Example 3: Analytical Sensitivity (Detection Limit)

The study was conducted to determine the analytical limit of detection (LoD) of the Xpert Flu/RSV XC assay using two batches of reagents across three test days. The maximum LoD observed for each strain and each batch was selected for validation. The validation required for the estimated LoD was performed for a reagent batch across a minimum of three test days. Two influenza A H3N2 strains, two influenza A 2009 H1N1 strains, two influenza B strains, two respiratory syncytial virus A (RSV A) strains, and two respiratory syncytial virus B (RSV B) strains diluted in a clinical matrix of negative collection were used to establish the LoD. The LoD was defined as the lowest concentration (tissue culture infectious dose, TCID50/mL) of each sample that could be repeatedly distinguished from the negative sample with 95% confidence, or the lowest concentration at which 19 out of 20 replicates were positive. Each virus concentration was tested with 20 replicates for each strain.

The LoD was determined empirically as the first concentration with 19/20 or 20/20 positive results. The LoD point values for each test strain are summarized in Tables 6 to 11.

Table 6. Confirmed LoD (TCID ₅₀ /mL): Influenza A 2009 H1N1

Table 7. Confirmed LoD (TCID ₅₀ /mL): Influenza A H3N2

Table 8. Confirmed LoD (TCID ₅₀ /mL): Influenza B

Table 9. Confirmed LoD (TCID ₅₀ /mL): Respiratory Syncytial Virus A

Table 10. Confirmed LoD (TCID ₅₀ /mL): Respiratory Syncytial Virus B

Table 11. Confirmed LoD (TCID ₅₀ /mL): Influenza A H7N9

Although the test has been shown to detect novel Avian Influenza A(H7N9) culture material, the performance characteristics of the device on clinical specimens positive for the novel Avian Influenza A(H7N9) virus have not been established. The Xpert Flu/RSV assay can differentiate between influenza A and influenza B viruses, but it cannot differentiate between influenza subtypes.

5.4. Example 4: Analytical Specificity (Exclusivity)

The analytical specificity of the Xpert Flu/RSV XC assay was evaluated by testing a panel of 44 cultures (consisting of 16 viruses, 26 bacteria, and two yeasts, representing common respiratory pathogens or those likely to be encountered in the nasopharynx). All bacterial and yeast strains were tested in triplicate at a concentration of ≥10 ⁶ CFU/mL. Triplicate cultures of all viruses were tested at a concentration of ≥10 ⁵ TCID50/mL. Analytical specificity was 100%. The results are shown in Table 12.

Table 12. Analytical Specificity of the Xpert Flu/RSV XC Assay

^a For A. baumannii, after initial testing, 1/3 replicates were positive for influenza A with a Ct of 39.2 (cutoff = 40). An additional 23 replicates were tested at >1 x 10 ⁶ CFU/mL; 23/23 replicates were correctly reported as positive for influenza A; negative for influenza B; and negative for RSV.

5.5 Example 5: Analysis of Reactivity (Inclusion)

The analytical reactivity of the Xpert Flu/RSV XC assay was evaluated at a level close to the analytical LoD for multiple influenza A (H1N1) strains (seasonal before 2009), influenza A (H1N1) (pandemic in 2009), influenza A (H3N2) (seasonal), avian influenza A (H5N1, H5N2, H6N2, H7N2, H7N3, H2N2, and H7N9), and H9N2), influenza B (representative strains from the Victoria and Yamagata lineages), and respiratory syncytial virus A/B (RSV A and RSV B). A total of 64 strains (comprising 54 influenza viruses and 10 RSV viruses) were tested using the Xpert Flu/RSV XC assay in this study.

Three replicates were tested for each strain. The results are shown in Table 13.

Table 13. Analytical Reactivity (Inclusive) of the Xpert Flu/RSV XC Assay

Table 13. Analytical Reactivity of the Xpert Flu/RSV XC Assay (Inclusive) (Continued)

Table 13. Analytical Reactivity of the Xpert Flu/RSV XC Assay (Inclusive) (Continued)

a Influenza A/Washington/24/2012 was tested at 5×LoD (80.0 TCID ₅₀ /mL) to obtain 3/3 replicates of influenza A positive result calls.

b Due to biosafety controls, viral RNA purified in simulated background matrix was used for avian influenza A virus.

c Due to biosafety control, inactivated avian influenza A (H7N9) virus with no viral titer was diluted 100,000-fold in a simulated background matrix and tested.

d Known Victoria lineage.

e Influenza B/Panama/45/90 was tested at 5×LoD (3.0 TCID ₅₀ /mL) to obtain 3/3 replicates of influenza B positive result calls.

f Known Yamagata lineage.

g RSV-B/CH93(18)-18 was tested at 10×LoD (20.0 TCID ₅₀ /mL) to obtain 3/3 replicates of RSV positive result calls.

5.6. Example 6: Interfering substances

In nonclinical studies, potential interfering substances that may be present in the nasopharynx were evaluated directly relative to the performance of the Xpert Flu/RSV XC assay. Potential interfering substances in the nasopharynx can include, but are not limited to: blood, nasal secretions or mucus, and nasal and throat medications used to relieve congestion, dry nose, irritation, or asthma and allergy symptoms, as well as antibiotics and antivirals. Negative samples (n=8) were tested for each substance to determine the impact on the performance of the sample processing control (SPC). Six influenza (four influenza A and two influenza B) and four RSV (two RSV A and two RSV B) strains were spiked at 2X the analytical LoD determined for each strain, and positive samples (n=8) were tested for each substance. All results were compared to positive and negative universal transport medium (UTM) controls.

The substances evaluated are listed in Table 14, showing the active ingredients and concentrations tested. No assay interference was observed in the presence of the substances at the concentrations tested in this study. All positive and negative replicates were correctly identified using the Xpert Flu/RSV XC assay. FluMist vaccine samples were correctly reported as positive for influenza A; positive for influenza; and negative for RSV, as expected. Samples containing FluMist may cause false positive results.

Table 14. Potential interfering substances in the Xpert Flu/RSV XC assay

5.7. Example 7: Carry-over contamination study

A study was conducted to show that a single-use, self-contained GeneXpert cartridge prevents carryover contamination in negative samples run after an extremely high positive sample in the same GeneXpert module. The study consisted of negative samples processed in the same GeneXpert module immediately following an extremely high influenza A sample (approximately 10 ⁶ TCID50/test). The test protocol was repeated 20 times on four GeneXpert modules for a total of 41 runs, resulting in 20 positive and 21 negative samples. All 20 positive samples were correctly reported as influenza A positive; influenza B negative; RSV negative. All 21 negative samples were correctly reported as influenza A negative; influenza B negative; RSV negative.

5.8. Example 8: Equivalence Study of Fresh vs. Frozen Samples

The equivalence of fresh and frozen (≥-70°C) samples in the Xpert Flu/RSV XC assay was evaluated by testing three different concentrations of individual influenza and RSV strains representing low positivity (2X LoD), moderate positivity (5X LoD), and high positivity (10X LoD) in a simulated background matrix. Negative samples consisted solely of simulated background matrix. Equivalence of fresh and frozen samples was determined using a seasonal influenza A H3N2 strain (A/Victoria/361/2011), an influenza B strain (B/Wisconsin/01/11), an RSV A strain (RSV A/Long/MD/56), and an RSV B strain (RSV B/9320/MA/77). For each sample type and concentration, 20 replicates were tested. All positive and negative samples were tested fresh, after one freeze-thaw cycle, and after two freeze-thaw cycles.

There was no difference in XpertFlu/RSV assay performance between fresh virus dilutions and two sequential freeze-thaw cycles for both positive and negative samples. All positive and negative replicates were correctly identified using the Xpert Flu/RSV XC assay.

5.9. Example 9: Reproducibility

A set of 10 samples with varying concentrations of influenza A, influenza B, and RSV were tested by two different operators at each of three locations on ten different days (10 samples x 1 time/day x 10 days x 2 operators x 3 locations). One batch of Xpert Flu/RSV XC assay cartridges was used at each of the three test locations. The Xpert Flu/RSV XC assay was performed according to the Xpert Flu/RSV XC assay procedure. The results are summarized in Table 15.

The reproducibility of the Xpert Flu/RSV XC assay was also evaluated with respect to the fluorescent signal expressed as Ct values for each detected target. The mean, standard deviation (SD), and coefficient of variation (CV) between sites, between days, between operators, and within the assay for each group member are provided in Table 16.

5.10. Example 10: Expected Assay Coverage

To determine expected influenza strain coverage, the Xpert Flu/RSV assay was used to sequence influenza isolates from January 1, 2009, to May 21, 2014, to identify mismatches in the primer and probe sequences in Table A. It was assumed that strains with mismatches in any of the primers and probes would affect the sensitivity of the assay, but would have a higher chance of being detected due to the increased redundancy of multiple primer and probe sequences for each influenza type. The impact of mismatches on assay sensitivity depended on the identity and position of the mismatch. Table 15 shows the results of the analysis.

As shown in Table 15, testing MP alone resulted in the failure to detect 1.8% of influenza A (H1N1) pdm09 isolates, 1.2% of influenza A (H1N1) seasonal isolates, and 0.6% of influenza A (H3N2) isolates. Adding PA and PB2 to the assay resulted in the detection of 100% of the influenza A isolates analyzed, suggesting that influenza isolates with mismatches relative to the MP primer and probe sequences did not have mismatches in at least one of the PA or PB2 primer and probe sequences. In addition, the Xpert Flu/RSV assay detected 98.5% of influenza B isolates, 96.2% of avian influenza H5 isolates, and 100% of avian influenza H7 isolates (or 98.4% of avian influenza H5+H7 isolates).

The data are summarized in Table 16 below:

Table 16: Summary of expected assay coverage

All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described, it will be understood that changes can be made without departing from the spirit and scope of the invention.

Tables of certain sequences

Sequence Listing

<110> CEPHEID

<120> Methods for detecting influenza

<130> CEPHD-33847/WO-1/PRI

<160> 43

<170> PatentIn version 3.5

<210> 1

<211> 3151

<212> DNA

<213> Influenza A

<400> 1

atggagagaa taaaagaact aagagatcta atgtcgcagt ctcgcactcg cgagatactc 60

actaagacca ctgtggacca tatggccata atcaaaaagt acacgtcagg aaggcaggag 120

aagaaccccg cactcagaat gaaatggatg atggcaatga aatacccaat tacagcagac 180

aggagaataa tggacatgat tccagagagg aatgaacaag gacaaaccct ctggagcaaa 240

acaaccgatg ctggatcgga ccgtgtgatg gtatcacccc tggccgtaac atggtggaat 300

aggaatggcc caacaacaag cacagttcac taccctaagg tatacaaaac ttatttcgaa 360

aaagtcgaaa ggttaaaaca tggtaccttt ggccctgtcc acttcagaaa tcaagttaaa 420

ataagaagga gggttgacac aaaccccggt catgcagatc tcagtgccaa ggaggcacag 480

gatgtgatca tggaagttgt tttcccaaac gaagtggggg caagaatact gacatcagag 540

tcacagctga caataacaaa agaaaagaaa gaagagctcc aggattgtaa aattgctccc 600

ttgatggtgg catacatgct agaaagagaa ttggttcgta agacgaggtt tcttccggtg 660

gctggtggaa caagcagtgt ttatattgaa gtgctgcact taactcaggg aacatgttgg 720

gaacaaatgt acactccagg aggagaagtg agaaatgatg atgttgacca aagtttgatt 780

atcgccgcta gaaacatagt aagaagagca gcagtgtcag cagacccatt agcatctctc 840

ttggaaatgt gccacagcac acaaattgga gatggagaga ataaaagaac taagagatct 900

aatgtcgcag tctcgcactc gcgagatact cactaagacc actgtggacc atatggccat 960

aatcaaaaag tacacgtcag gaaggcagga gaagaacccc gcactcagaa tgaaatggat 1020

gatggcaatg aaatacccaa ttacagcaga caggagaata atggacatga ttccagagag 1080

gaatgaacaa ggacaaaccc tctggagcaa aacaaccgat gctggatcgg accgtgtgat 1140

ggtatcaccc ctggccgtaa catggtggaa taggaatggc ccaacaacaa gcacagttca 1200

ctaccctaag gtatacaaaa cttatttcga aaaagtcgaa aggttaaaac atggtacctt 1260

tggccctgtc cacttcagaa atcaagttaa aataagaagg agggttgaca caaaccccgg 1320

tcatgcagat ctcagtgcca aggaggcaca ggatgtgatc atggaagttg ttttcccaaa 1380

cgaagtgggg gcaagaatac tgacatcaga gtcacagctg acaataacaa aagaaaagaa 1440

agaagagctc caggattgta aaattgctcc cttgatggtg gcatacatgc tagaaagaga 1500

attggttcgt aagacgaggt ttcttccggt ggctggtgga acaagcagtg tttatattga 1560

agtgctgcac ttaactcagg gaacatgttg ggaacaaatg tacactccag gaggagaagt 1620

gagaaatgat gatgttgacc aaagtttgat tatcgccgct agaaacatag taagaagagc 1680

agcagtgtca gcagacccat tagcatctct cttggaaatg tgccacagca cacaaattgg 1740

aggaataagg atgatggaca tccttagaca gaacccaacg gaggaacaag ccgtagacat 1800

atgcaaggca gcaatggggc tgaggattag ctcctctttc agctttggtg ggttcacctt 1860

caaaaggaca agcggatcat ctgttaagaa agaagaagaa gtgctcacgg gcaacctcca 1920

aacactgaaa ataagagtac atgaaggata tgaggaattc acaatggtcg ggagaagagc 1980

aacagctatt ctcagaaaag caaccaggag attgatccag ttaatagtaa gtggaagaga 2040

cgatcaatca attgctgagg caataattgt ggccatggta ttttcacaag aggattgcat 2100

gatcaaagca gttaggggcg atctgaactt tgtcaatagg gcaaaccagc gactgaatcc 2160

catgcaccaa ctcttgaggc atttccaaaa ggatgcaaaa gtgcttttcc agaactgggg 2220

gattgaaccc atcgacagtg taatgggaat gatcggaata ttgcctgata tgaccccaag 2280

cacggaaatg tcactgagag gtataagagt cagcaaaatg ggagtagatg aatattccag 2340

tacggagaga gtggtagtga gcattgaccg atttttgaga gttcggggatc aacgagggaa 2400

cgtactattg tcccccgaag aggtcagcga gacacaggga actgagaaat tgaccataac 2460

ttattcgtca tcaatgatgt gggagatcaa tggtcctgag tcagtgctgg tcaacactta 2520

tcaatggatc ataaggaact gggaaagctt gaaaattcaa tggtcacagg atcccacgat 2580

gttatacaac aaaatggaat ttgaaccatt ccagtctctt gtccctaagg caaccagaag 2640

tcgttacagt ggattcgtga ggacactgtt ccagcaaatg cgggatgtgc ttggaacatt 2700

tgatactgtc caaataataa agcttctccc ctttgctgca gctccaccgg aacagagtag 2760

gatgcagttc tcctcgctga ctgtgaatgt aagaggatca gggctgagga tactggtaag 2820

aggcaattct ccagtgttca attacaataa agcaaccaaa aggcttacaa ttcttggaaa 2880

agatgcaggt gcattgactg aagatccaga tgaaggcaca gctggagtgg agtctgctgt 2940

cctgagggga ttcctcattt tgggtaaaga agacaagaga tatggcccag cattaagcat 3000

caatgaactg agcaatcttg caaaaggaga gaaggctaat gtgctaattg ggcaaggaga 3060

cgtggtgttg gtaatgaaac ggaaacggga ctctagcata cttactgaca gccagacagc 3120

gaccaaaagg attcggatgg ccatcaatta g 3151

<210> 2

<211> 2587

<212> DNA

<213> Influenza A

<400> 2

atggaagact ttgtgcgaca atgcttcaat ccgatgatcg tcgagcttgc ggaaaaggca 60

atgaaagaat atggggaaga tccgaaaatc gaaactaaca agtttgctgc aatatgcaca 120

catttggaag tttgtttcat gtattcggat ttccatttca tcgacgaacg gggtgaatca 180

ataattgtag aatctggtga cccgaatgca ctattgaagc accgatttga gataattgaa 240

ggaagagacc gaatcatggc ctggacagtg gtgaacagta tatgtaacac aacaggggta 300

gagaagccta aatttcttcc tgatttgtat gattacaaag aaaaccggtt cattgaaatt 360

ggagtaacac ggagggaagt ccacatatat tacctagaga aagccaacaa aataaaatct 420

gagaagacac acattcatgg aagactttgt gcgacaatgc ttcaatccga tgatcgtcga 480

gcttgcggaa aaggcaatga aagaatatgg ggaagatccg aaaatcgaaa ctaacaagtt 540

tgctgcaata tgcacacatt tggaagtttg tttcatgtat tcggatttcc atttcatcga 600

cgaacggggt gaatcaataa ttgtagaatc tggtgacccg aatgcactat tgaagcaccg 660

atttgagata attgaaggaa gagaccgaat catggcctgg acagtggtga acagtatatg 720

taacacaaca ggggtagaga agcctaaatt tcttcctgat ttgtatgatt acaaagaaaa 780

ccggttcatt gaaattggag taacacggag ggaagtccac atatattacc tagagaaagc 840

caacaaaata aaatctgaga agacacacat tcacatcttt tcattcactg gagaggagat 900

ggccaccaaa gcagactaca cccttgacga agagagcagg gcaagaatca aaactaggct 960

tttcactata agacaagaaa tggccagtag gagtctatgg gattcctttc gtcaatccga 1020

aagaggcgaa gagacaattg aagaaaaatt tgagattaca ggaactatgc gcaagcttgc 1080

cgaccaaagt ctcccaccga acttctccag ccttgaaaac tttagagcct atgtagatgg 1140

attcgagccg aacggctgca ttgagggcaa gctttcccaa atgtcaaagg aagtgaacgc 1200

caaaattgaa ccattcttga ggacgacacc acgccccctc agattgcctg atgggcctct 1260

ttgccatcag cggtcaaagt tcctgctgat ggatgctctg aaattaagta ttgaagaccc 1320

gagtcacgag ggagagggaa taccactata tgatgcaatc aaatgcatga agacattctt 1380

tggctggaaa gagcctaaca tagtcaaacc acataagaaa ggcataaatc ccaattacct 1440

tatggcttgg aagcaggtgc taacagagct acaggacatt gaaaatgaag agaagatccc 1500

aaggacaaag aacatgaaga gaacaagcca attgaagtgg gcactcggtg aaaatatggc 1560

accagaaaaa gtagactttg atgactgcaa agatgttgga gaccttaaac agtatgacag 1620

tgatgagcca gagcccagat ctctagcaag ctgggtccaa aatgaattca ataaggcatg 1680

tgaattgact gattcaagct ggatagaact tgatgaaata ggagaagatg ttgccccgat 1740

tgaacatatc gcaagcatga ggaggaacta ttttacagca gaagtgtccc actgcagggc 1800

tactgaatac ataatgaagg gagtgtacat aaatacggcc ttgctcaatg catcctgtgc 1860

agccatggat gactttcagc tgatcccaat gataagcaaa tgtaggacca aagaaggaag 1920

acggaaaaca aacctgtatg ggttcattat aaaaggaagg tctcatttga gaaatgatac 1980

tgatgtggtg aactttgtaa gtatggagtt ctcactcact gacccgagac tggagccaca 2040

caaatgggaa aaatactgtg ttcttgaaat aggagacatg ctcttgagga ctgcgatagg 2100

ccaagtgtcg aggcccatgt tcctatatgt gagaaccaat ggaacctcca agatcaagat 2160

gaaatggggc atggaaatga ggcgctgcct tcttcagtcc cttcagcaga ttgagagcat 2220

gattgaggcc gagtcttctg tcaaagagaa agacatgacc aaggaattct ttgaaaacaa 2280

atcagaaaca tggccaatcg gagagtcacc cagaggagtg gaggaaggct ctattgggaa 2340

agtgtgcagg accttactgg caaaatctgt gttcaacagt ctatatgcgt ctccacaact 2400

tgaggggttt tcggctgaat cgagaaaatt gcttctcatt gttcaggcac ttagggacaa 2460

cctggaacct ggaaccttcg atcttggggg gctatatgaa gcaatcgagg agtgcctgat 2520

taatgatccc tgggttttgc ttaatgcatc ttggttcaac tccttcctca cacatgcact 2580

gaagtag 2587

<210> 3

<211> 982

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

atgagtcttc taaccgaggt cgaaacgtac gttctttcta tcataccgtc aggccccctc 60

aaagccgaga tcgcgcagag actggaaagt gtctttgcag gaaagaacac agatcttgag 120

gctctcatgg aatggctaaa gacaagacca atcttgtcac ctttgactaa gggaatttta 180

ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240

caaaatgccc taaatgggaa tggggaccca aacaacatgg atagagcagt taaactatac 300

aagaagctca aaagagaaat aacgttccat ggggccaagg aggtgtcact aagctattca 360

actggtgcac ttgccagttg catgggcctc atatacaaca ggatgggaac agtgaccaca 420

gaagctgcttttggtctagt gtgtgccact tgtgaacaga ttgctgattc acagcatcgg 480

tctcacagac agatggctac taccaccaat ccactaatca ggcatgagaa cagaatggtg 540

ctggctagca ctacggcaaa ggctatggaa cagatggctg gatcgagtga acaggcagcg 600

gaggccatgg aggttgctaa tcagactagg cagatggtac atgcaatgag aactattggg 660

actcatccta gctccagtac tggtctgaaa gatgaccttc ttgaaaattt gcaggcctac 720

cagaagcgaa tggggagtgca gatgcagcga ttcaagtgat cctctcgcca ttgcagcaaa 780

tatcattggg atcttgcacc tgatattgtg gattactgat cgtctttttt tcaaatgtat 840

ttatcgtcgc tttaaatacg gtttgaaaag agggccttct acagaaggag tgcctgagtc 900

catgagggaa gaatatcaac aggaacagca gagtgctgtg gatgttgacg atggtcattt 960

tgtcaacata gagctagagt aa 982

<210> 4

<211> 931

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

cgtacgttct atctatcatt ccatcaggcc ccctcaaagc cgagatcgcg cagagacttg 60

aggatgtttt tgcagggaag aacgcagatc tcgaggctct catggagtgg ataaagacaa 120

gaccaatcct gtcacctctg actaagggga ttttagggtt tgtgttcacg ctcaccgtgc 180

ccagtgagcg aggactgcag cgtagacggt ttgtccaaaa cgccctaaat gggaatggag 240

acccaaacaa catggacaag gcagttaaat tatacaagaa actgaagagg gaaatgacat 300

tccatggagc aaaggaagtt gcactcagtt actcaactgg tgcgcttgcc agctgcatgg 360

gtctcatata caacaggatg gggacagtaa ctgcagaagg ggctcttgga ttggtatgtg 420

ccacttgtga gcagattgct gacgcacaac atcggtccca caggcagatg gcaactacta 480

ccaacccact aattaggcat gagaatagaa tggtactagc cagtactacg gctaaggcta 540

tggagcagat ggctggatca agtgaacagg cagcggaagc catggaagtt gcaagccagg 600

ctaggcaaat ggtgcaggct atgagaacag tcgggactca ccctaactcc agtacaggtc 660

taaaggatga tcttattgaa aatttgcagg cttaccagaa ccggatggga gtgcaactgc 720

agcggttcaa gtgatcctct cgttgttgca gctaacatta ttgggatatt gcacttgata 780

ttgtggattc ttgatcgtct tttcttcaaa tgcatttatc gtcgctttaa atacggtttg 840

aaaagagggc cttctacgga aggaatgcct gagtctatga gggagaata tcggcaggaa 900

cagcagaatg ctgtggatgt tgacgatggt c 931

<210> 5

<211> 1683

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

atgaacactc aaatcctggt attcgctctg attgcgatca ttccaacaaa tgcagacaaa 60

atctgcctcg gacatcatgc cgtgtcaaac ggaaccaaag taaacacatt aactgaaaga 120

ggagtggaag tcgtcaatgc aactgaaaca gtggaacgaa caaacatccc caggatctgc 180

tcaaaaggga aaatgacagt tgacctcggt caatgtggac tcctggggac aatcactgga 240

ccacctcaat gtgaccaatt cctagaattt tcagccgatt taattattga gaggcgagaa 300

ggaagtgatg tctgttatcc tgggaaattc gtgaatgagg aagctctgag gcaaatactc 360

agagaatcag gcggaattga caaggaagca atgggattca catacagtgg aataagaact 420

aatggagcaa ccagtgcatg taggagatca ggatcttcat tctatgcaga aatgaaatgg 480

ctcctgtcaa acacagataa tgctgcattc ccgcagatga ctaagtcata taaaaataca 540

agaaaaagcc cagctctaat agtatggggg atccatcatt ccgtatcaac tgcagagcaa 600

accaagctat atgggagtgg aaacaaactg gtgacagttg ggagttctaa ttatcaacaa 660

tcttttgtac cgagtccagg agcgagacca caagttaatg gtctatctgg aagaattgac 720

tttcattggc taatgctaaa tcccaatgat acagtcactt tcagtttcaa tggggctttc 780

atagctccag accgtgcaag cttcctgaga ggaaaatcta tgggaatcca gagtggagta 840

caggttgatg ccaattgtga aggggactgc tatcatagtg gagggacaat aataagtaac 900

ttgccatttc agaacataga tagcagggca gttggaaaat gtccgagata tgttaagcaa 960

aggagtctgc tgctagcaac agggatgaag aatgttcctg agattccaaa gggaagaggc 1020

ctatttggtg ctatagcggg tttcattgaa aatggatggg aaggcctaat tgatggttgg 1080

tatggtttca gacaccagaa tgcacaggga gagggaactg ctgcagatta caaaagcact 1140

caatcggcaa ttgatcaaat aacaggaaaa ttaaaccggc ttatagaaaa aaccaaccaa 1200

caatttgagt tgatagacaa tgaattcaat gaggtagaga agcaaatcgg taatgtgata 1260

aattggacca gagattctat aacagaagtg tggtcataca atgctgaact cttggtagca 1320

atggagaacc agcatacaat tgatctggct gattcagaaa tggacaaact gtacgaacga 1380

gtgaaaagac agctgagaga gaatgctgaa gaagatggca ctggttgctt tgaaatattt 1440

cacaagtgtg atgatgactg tatggccagt attagaaata acacctatga tcacagcaaa 1500

tacagggaag aggcaatgca aaatagaata cagattgacc cagtcaaact aagcagcggc 1560

tacaaagatg tgatactttg gtttagcttc ggggcatcat gtttcatact tctagccatt 1620

gtaatgggcc ttgtcttcat atgtgtaaag aatggaaaca tgcggtgcac tatttgtata 1680

taa 1683

<210> 6

<211> 1139

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

atgtcgctgt ttggagacac aattgcctac ctgctttcat tgacagagga tggagaaggc 60

aaagcagaac tagcagaaaa attacactgt tggtttggtg ggaaagaatt tgacctagac 120

tctgccttgg aatggataaa aaacaaaaga tgcttaactg atatacaaaa agcactaatt 180

ggtgcctcta tatgcttttt aaaacccaaa gaccaggaaa gaaaaagaag attcatcaca 240

gagcccttat caggaatggg aacaacagca acaaaaaaga aaggcctgat tctggctgag 300

agaaaaatga gaagatgtgt gagctttcat gaagcatttg aaatagcaga aggccatgaa 360

agctcagcgc tactatactg tctcatggtc atgtacctga atcctggaaa ttattcaatg 420

caagtaaaac taggaacgct ctgtgcttta tgcgagaaac aagcatcaca ttcacacagg 480

gctcatagca gagcagcgag atcttcagtg cctggagtga gacgagaaat gcagatggtc 540

tcagctatga acacagcaaa aacaatgaat ggaatgggaa aaggagaaga cgtccaaaag 600

ctggcagaag agttgcaaag caacattgga gtgctgagat ctcttggagc aagccaaaag 660

aatggggaag ggattgcaaa ggatgtaatg gaagtgctaa agcagagctc catgggaaat 720

tcagctcttg tgaagaaata tctataatgc tcgaaccatt tcagattctt acaatttgtt 780

cttttatctt atcagctctc catttcatgg cttggacaat agggcatttg aatcaaataa 840

aaagaggaat aaacatgaaa atacgaataa aaggtccaaa caaagagaca ataaacagag 900

aggtatcaat tttgagacac agttaccaaa aagaaatcca ggccaaagaa acaatgaagg 960

aagtactctc tgacaacatg gaggtattga atgaccacat aataattgag gggctttctg 1020

ccgaagagat aataaaaatg ggtgaaacag ttttggagat agaagaattg cattaaattc 1080

aatttacta tatttcttac tatgcattta agcaaattgt aatcaatgtc agcaaataa 1139

<210> 7

<211> 1054

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

atggcgaaca acaacatgac cacaacacaa attgaggtgg gtccgggagc aaccaatgcc 60

accataaact ttgaagcagg aattctggag tgctatgaaa ggctttcatg gcaaagagcc 120

cttgactacc ccggtcaaga ccgcctaaac agactaaaga gaaaattaga gtcaagaata 180

aagactcaca acaaaagtga gcctgaaagt aaaaggatgt cccttgaaga gagaaaagca 240

attggagtaa aaatgatgaa agtactccta tttatgaatc cgtctgctgg aattgaaggg 300

tttgagccat actgtatgaa cagttcctca aatagcaact gtacgaaata caattggacc 360

gattaccctt caacaccaga gaggtgcctt gatgacatag aggaagaacc agaggatgtt 420

gatggcccaa ctgaaatagt attaagggac atgaacaaca aagatgcaag gcaaaagata 480

aaggaggaag taaacactca gaaagaaggg aagttccgtt tgacaataaa aagggatatg 540

cgtaatgtat tgtccttgag agtgttggta aatggaacat tcctcaaaca ccccaatgga 600

tacaagtcct tatcaactct gcatagattg aatgcatatg accagagtgg aaggcttgtt 660

gctaaacttg ttgccactga tgatcttaca gtggaggatg aagaagatgg ccatcggatc 720

ctcaactcac tcttcgagcg tcttaatgaa ggacattcaa agccaattcg agcagctgaa 780

actgcggtgg gagtcttatc ccaatttggt caagagcacc gattatcacc agaagaggga 840

gacaattaga ctggtcacgg aagaacttta tcttttaagt aaaagaattg atgataacat 900

actattccac aaaacagtga tagctaacag ctccataata gctgacatgg ttgtatcatt 960

atcattatta gaaacattgt atgaaatgaa ggatgtggtt gaagtgtaca gcaggcagtg 1020

cttgtgaatt taaaataaaa atcctgttac tact 1054

<210> 8

<211> 68

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

aaacgggact ctagcatact tactgacagc cagacagcga ccaaaaggat tcggatggcc 60

atcaatta 68

<210> 9

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

atcttggggg gctatatgaa gcaatcgagg agtgcctgat taatgatccc tgggttttgc 60

ttaatgcatc ttggttcaac tccttcct 88

<210> 10

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

ttctaaccga ggtcgaaacg tacgttcttt ctatcatacc gtcaggcccc ctcaaagccg 60

agatcgcgca gagactggaa agtgtctttg caggaaagaa cacagatctt gaggctctca 120

tggaatggct aaagacaaga ccaat 145

<210> 11

<211> 91

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

caagaccaat cctgtcacct ctgactaagg ggattttagg gtttgtgttc acgctcaccg 60

tgccccagtga gcgaggactg cagcgtagac g 91

<210> 12

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

gaaatgaaat ggctcctgtc aaacacagat aatgctgcat tcccgcagat gactaagtca 60

tataaaaata caagaaaaag c 81

<210> 13

<211> 107

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

tttggagaca caattgccta cctgctttca ttgacagagg atggagaagg caaagcagaa 60

ctagcagaaa aattacactg ttggtttggt gggaaagaat ttgacct 107

<210> 14

<211> 112

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

gatggccatc ggatcctcaa ctcactcttc gagcgtctta atgaaggaca ttcaaagcca 60

attcgagcag ctgaaactgc ggtggggagtc ttatcccaat ttggtcaaga gc 112

<210> 15

<211> 121

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

tacactcaac aaagatcaac ttctgtcatc cagcaaatac accatccaac ggagcacagg 60

agatagtatt gatactccta attatgatgt gcagaaacac atcaacaagt tatgtggcat 120

g 121

<210> 16

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

cattaaataa ggatcagctg ctgtcatcca gcaaataacac tattcaacgt agtacaggag 60

ataatattga cactcccaat tatgatgtgc aaaaacacct aaacaaacta tgtggtatgc 120

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

aaacgggact ctagcatact 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

taattgatgg ccatccgaat 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

agccagacag cgaccaaaag 20

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

atcttggggg gctatatgaa gcaat 25

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

aggaaggagt tgaaccaaga 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

aatgatccct gggttttgct 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

ttctaaccga ggtcgaaacg 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

attggtcttg tctttagcca 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

tcaggccccc tcaaagccga 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

caagaccaat cctgtcacct 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

cgtctacgct gcagtcctcg 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

acgctcaccg tgcccagtga 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 29

gaaatgaaat ggctcctgtc 20

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 30

ggctttttct tgtattttta tatga 25

<210> 31

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 31

ctgcattccc gcagatgac 19

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

ttggagacac gattgcctac 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

aggtcaaatt ctttcccacc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

atggagaagg caaagcagaa 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

gatggccatc ggatcctcaa 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

gctcttgacc aaattgggat 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

aaagccaatt cgagcagctg 20

<210> 38

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

tacactcaac aaagatcaac ttctgtc 27

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

catgccacat aacttattga tgtgt 25

<210> 40

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

caccatccaa cggagcacag gaga 24

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 41

cattaaataa ggatcagctg ctgtc 25

<210> 42

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

gcataccaca tagtttgttt aggtgtt 27

<210> 43

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

taatattgac actcccaatt atgatgtgc 29

Claims (48)

1. Use of a reagent for detecting the presence or absence of at least one influenza gene selected from acid polymerase (PA) and alkaline polymerase 2 (PB2) in a sample in the preparation of a kit or composition for detecting the presence or absence of influenza in a sample from a subject, wherein the reagent comprises a first primer pair and a first probe for acid polymerase (PA), wherein the first probe is for contacting an amplicon generated by the first primer pair, wherein the first primer pair comprises a first primer and a second primer, and wherein the first primer comprises the sequence of SEQ ID NO:20, and the second primer comprises the sequence of SEQ ID NO:21. 2. The use according to claim 1, wherein the presence of at least one influenza gene in the sample indicates that the subject has influenza. 3. The use according to claim 1, wherein the PA gene and/or PB2 gene are influenza A genes. 4. The use according to claim 1, wherein the sequence of the PA gene is identical to the sequence of SEQ ID NO: 2. 5. The use according to claim 1, wherein the sequence of the PB2 gene is identical to the sequence of SEQ ID NO: 1. 6. The use according to claim 1, wherein the kit or composition further comprises a reagent for detecting the presence or absence of at least one influenza matrix protein (MP) gene, wherein the MP gene is an influenza A MP gene, an influenza B MP gene, and/or an avian influenza MP gene. 7. The use according to claim 6, wherein the avian influenza MP gene is a hemagglutinin (H)5 or H7 subtype. 8. The use according to claim 6, wherein the sequence of the influenza A MP gene is identical to the sequence of SEQ ID NO: 3 or 4, and the sequence of the influenza B MP gene is identical to the sequence of SEQ ID NO: 6. 9. The use according to claim 1, wherein the kit or composition further comprises a reagent for detecting the presence or absence of at least one influenza non-structural (NS) gene. 10. The use according to claim 9, wherein the NS gene is the influenza B NS gene, and wherein the sequence of the influenza B NS gene is identical to the sequence of SEQ ID NO: 7. 11. The use according to claim 1, wherein the kit or composition further comprises a reagent for detecting the presence or absence of at least one influenza hemagglutinin (HA) gene, wherein the HA gene is the influenza A HA gene and/or the avian influenza HA gene. 12. The use according to claim 11, wherein the avian influenza is the H7 subtype. 13. The use according to claim 11, wherein the sequence of the influenza HA gene is identical to the sequence of SEQ ID NO: 5. 14. The use according to claim 1, wherein the kit or composition further comprises a reagent for detecting the presence or absence of the influenza A PA gene, influenza A PB2 gene, influenza A MP gene, avian influenza MP gene, and avian influenza HA gene, wherein the sequence of the influenza A PA gene is identical to SEQ ID NO: 2, the sequence of the influenza A PB2 gene is identical to SEQ ID NO: 1, the sequence of the influenza A MP gene is identical to SEQ ID NO: 3, the sequence of the avian influenza MP gene is identical to SEQ ID NO: 4, and the sequence of the avian influenza HA gene is identical to SEQ ID NO: 5. 15. The use according to claim 1, wherein the kit or composition further comprises reagents for detecting the presence or absence of the influenza B MP gene and the influenza B NS gene. 16. The use according to claim 15, wherein the sequence of the influenza B MP gene is identical to SEQ ID NO: 6 and the sequence of the influenza B NS gene is identical to SEQ ID NO: 7. 17. The use according to claim 1, wherein the kit or composition further comprises a reagent for detecting the presence or absence of respiratory syncytial virus (RSV) in a sample from the subject. 18. The use according to claim 1, wherein the kit or composition comprises a reagent for detecting an exogenous control. 19. The use according to claim 1, wherein the reagent for detecting the influenza PB2 gene is a second primer pair. 20. The use of claim 19, wherein the second primer pair comprises a third primer and a fourth primer, wherein the third primer consists of the sequence of SEQ ID NO: 17, and the fourth primer consists of the sequence of SEQ ID NO: 18. 21. The use of claim 19, wherein the kit or composition further comprises at least one additional primer pair, wherein each of the additional primer pairs is used to detect different influenza genes selected from the influenza A MP gene, the avian influenza MP gene, and the avian influenza HA gene. 22. The use according to claim 21, wherein each additional primer pair comprises a fifth primer and a sixth primer, said fifth primer and sixth primer being independently selected from: a) The fifth primer consists of the sequence of SEQ ID NO:23, and the sixth primer consists of the sequence of SEQ ID NO:24; b) The fifth primer consists of the sequence of SEQ ID NO:26, and the sixth primer consists of the sequence of SEQ ID NO:27; c) The fifth primer consists of the sequence of SEQ ID NO:29, and the sixth primer consists of the sequence of SEQ ID NO:30. 23. The use of claim 19, wherein the kit or composition further comprises at least one additional primer pair, wherein each additional primer pair is used to detect different influenza genes selected from the influenza B MP gene and the influenza B NS gene. 24. The use as claimed in claim 23, wherein each additional primer pair comprises a seventh primer and an eighth primer, said seventh primer and eighth primer being independently selected from: a) The seventh primer consists of the sequence of SEQ ID NO:32 and the sequence of the eighth primer SEQ ID NO:33; b) The seventh primer consists of the sequence of SEQ ID NO:35, and the eighth primer consists of the sequence of SEQ ID NO:36. 25. The use of claim 19, wherein the composition or kit further comprises at least one additional primer pair, wherein each additional primer pair is used to detect RSV A or RSV B. 26. The use as claimed in claim 25, wherein each additional primer pair comprises a ninth primer and a tenth primer, said ninth primer and tenth primer being independently selected from: a) The ninth primer consists of the sequence of SEQ ID NO:38, and the tenth primer consists of the sequence of SEQ ID NO:39; b) The ninth primer consists of the sequence of SEQ ID NO:41, and the tenth primer consists of the sequence of SEQ ID NO:42. 27. The use according to claim 1, wherein the kit or composition comprises primer pairs for detecting the influenza A PA gene, influenza A PB2 gene, influenza A MP gene, avian influenza MP gene, avian influenza HA gene, and optionally primer pairs for detecting the influenza B MP gene and/or influenza B NS gene, and optionally primer pairs for detecting RSV A and/or RSV B. 28. The use of claim 27, wherein the kit or composition further comprises a control primer pair for detecting an exogenous control. 29. The use as claimed in claim 27, wherein each primer pair produces an amplicon that is 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, or 50 to 150 nucleotides long. 30. The use of claim 29, wherein at least one amplicon is selected from influenza A PA amplicon and influenza A PB2 amplicon, wherein the influenza A PA amplicon contains the sequence of SEQ ID NO: 9 and the influenza A PB2 amplicon contains the sequence of SEQ ID NO: 8. 31. The use according to claim 29, wherein at least one amplicon is selected from influenza A MP amplicon, avian influenza MP amplicon, and avian influenza HA amplicon, wherein the influenza A MP amplicon contains the sequence of SEQ ID NO: 10, the avian influenza MP amplicon contains the sequence of SEQ ID NO: 11, and the avian influenza HA amplicon contains the sequence of SEQ ID NO: 12. 32. The use of claim 29, wherein the amplicon is an influenza B MP amplicon and/or an influenza B NS amplicon, wherein the influenza B MP amplicon contains the sequence of SEQ ID NO: 13 and the influenza B NS amplicon contains the sequence of SEQ ID NO: 14. 33. The use of claim 29, wherein the amplicon is an RSV A amplicon and/or an RSV B amplicon, wherein the RSV A amplicon contains the sequence of SEQ ID NO: 15 and the RSV B amplicon contains the sequence of SEQ ID NO: 16. 34. The use of claim 30, wherein the kit or composition further comprises at least one probe selected from influenza A PA probe and influenza A PB2 probe, wherein each probe optionally contains a detectable marker, wherein the detectable marker may be the same or different. 35. The use according to claim 34, wherein the influenza PA probe consists of the sequence of SEQ ID NO: 22, and the influenza PB2 probe consists of the sequence of SEQ ID NO: 19. 36. The use of claim 31, wherein the kit or composition further comprises at least one probe contact selected from influenza A MP probe, avian influenza MP probe and avian influenza HA probe, wherein each probe optionally contains a detectable marker, wherein the detectable marker may be the same or different. 37. The use according to claim 36, wherein the influenza MP probe is composed of the sequence of SEQ ID NO: 25, the avian influenza MP probe is composed of the sequence of SEQ ID NO: 28, and the avian influenza HA probe is composed of the sequence of SEQ ID NO: 31. 38. The use of claim 32, wherein the kit or composition further comprises at least one probe selected from influenza B MP probes and influenza B NS probes, wherein each probe optionally contains a detectable marker, wherein the detectable marker may be the same or different. 39. The use according to claim 38, wherein the influenza B MP probe consists of the sequence of SEQ ID NO: 34, and the influenza B NS probe consists of the sequence of SEQ ID NO: 37. 40. The use of claim 33, wherein the kit or composition further comprises at least one probe selected from RSV A probes and RSV B probes, wherein each probe optionally contains a detectable marker, wherein the detectable marker may be the same or different. 41. The use of claim 40, wherein the RSV A probe comprises the sequence of SEQ ID NO: 40, and the RSV B probe comprises the sequence of SEQ ID NO: 43. 42. The use according to claim 1, wherein the sample is selected from nasopharyngeal swab samples, nasal aspirate samples, and nasal wash samples. 43. A composition comprising: (a) A first primer pair for detecting the influenza PA gene, wherein the first primer pair comprises a first primer and a second primer, wherein the first primer consists of the sequence of SEQ ID NO: 20; and wherein the second primer consists of the sequence of SEQ ID NO: 21; and (b) A second primer pair for detecting the influenza PB2 gene, wherein the second primer pair comprises a third primer and a fourth primer, wherein the third primer consists of the sequence of SEQ ID NO: 17, and wherein the fourth primer consists of the sequence of SEQ ID NO: 18; (c) A first probe of acid polymerase (PA), wherein the first probe is used to contact the amplicon generated by the first primer pair; and (d) A second probe of alkaline polymerase (PB2), wherein the second probe is used to contact the amplicon generated by the second primer pair. 44. The composition of claim 43, further comprising at least one additional primer pair, wherein each additional primer pair is used to detect different influenza genes selected from the influenza A MP gene, avian influenza MP gene, avian influenza HA gene, influenza B MP gene, influenza B NS gene, RSV A gene, and RSV B gene. 45. The composition of claim 43, comprising an influenza A PA probe and an influenza A PB2 probe. 46. The composition of claim 45, wherein the influenza A PA probe comprises the sequence of SEQ ID NO: 22, and the influenza PB2 probe comprises the sequence of SEQ ID NO: 19. 47. The composition of claim 45, further comprising at least one probe selected from influenza A MP probe, avian influenza MP probe, avian influenza HA probe, influenza B MP probe, influenza B NS probe, RSV A probe and RSV B probe, wherein each probe comprises a detectable marker. 48. A kit comprising the composition of claim 45, wherein the kit optionally further comprises an exogenous control.