JP7803127B2 - Respiratory syncytial virus detection probe and its use - Google Patents

Description

The present invention relates to a probe for detecting RS virus, a method for detecting RS virus using the probe, and reagents and kits for use in the method.

Respiratory tract infections caused by the respiratory syncytial virus (RSV) frequently cause bronchitis and pneumonia, especially in infants and the elderly who are infected for the first time. RSV has genomic RNA that encodes 11 types of proteins, and among these proteins, it is broadly classified into subgroups, type A and type B, based on differences in the properties of the G protein.

To date, genetic testing, antigen testing, and antibody testing have been developed as methods for testing for RS virus. Of these, antigen testing has the advantage of being simple, but currently suffers from low sensitivity. Furthermore, antibody testing can confirm whether a person has had the disease in the past, but is not suitable for testing whether they are currently infected. In contrast, genetic testing has the advantage of being able to specifically detect RS virus with excellent sensitivity.

The genetic testing method for RS virus involves first using reverse transcriptase (RT).
A typical detection method involves reverse transcription (RT) of RS virus genomic RNA into cDNA using ribosomal RNA cleavage enzyme (RIP), followed by nucleic acid amplification using PCR or other methods. Currently, the most commonly used RT-PCR detection method involves quantitative detection by performing real-time PCR using a double-labeled nucleic acid probe (also known as a Taqman probe, hydrolysis probe, etc.) (Non-Patent Document 1). While this method allows for the quantification of viral load based on control RNA, photometry is required for each PCR cycle, requiring approximately one hour for detection even at the shortest possible time.

Furthermore, melting curve analysis is known as a method for detecting nucleic acid amplification products. Melting curve analysis allows nucleic acid amplification and detection to be performed in separate steps, allowing for relatively simple measurements in as little as 30 minutes. Furthermore, melting curve analysis using fluorescently labeled nucleic acid probes has the advantage of being easily adaptable to genetic testing using automated analyzers. However, no nucleic acid probes capable of detecting RS virus with high sensitivity using melting curve analysis are known yet.

It is generally known that type A RS virus is more likely to cause severe illness. Subgroup types A and B are known to have partial differences in their base sequences, making it generally difficult to detect both types in the same reaction system. Furthermore, adding multiple primer-probe sets to the same reaction system increases the likelihood of inducing interactions, such as the formation of dimers between oligonucleotides, and finding the optimal primer and probe sequences requires extensive trial and error. Furthermore, RS virus can undergo mutations in the genomic RNA region. Therefore, it is not easy to design primers and probes that can detect these as comprehensively as possible.

National Institute of Infectious Diseases, Human Orthopneumovirus (RSV) Pathogen Detection Manual, Version 2.0 (June 12, 2020)

The present invention was made in response to these problems in the prior art. Specifically, the object of the present invention is to provide a probe useful for detecting RS virus and a method for detecting RS virus using the same.

As a result of extensive research to achieve the above objective, the inventors discovered that RS virus can be detected easily, quickly, and with high sensitivity by using a labeled probe with a specific base sequence. Based on this finding, the inventors conducted further extensive research and completed the present invention.

The present invention includes the following aspects.
[Item 1] A probe for detecting RS virus, having the following characteristics (A) and (B):
(A) containing the following base sequence (A-1) or (A-2):
(A-1) A base sequence of at least 10 consecutive bases in a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which contains at least a base sequence complementary to the 45th to 52nd bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or (A-2) A base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence of (A-1); and (B) Only either the 5' end or the 3' end is labeled.
[Item 2] The probe according to Item 1, wherein the length of the base sequence of (A) is 14 to 26 bases.
[Item 3] The probe according to Item 1 or 2, wherein the base sequence of (A) comprises a base sequence complementary to the base sequence shown in any one of SEQ ID NOs: 3 to 7.
[Item 4] The probe according to any one of Items 1 to 3, wherein the label (B) is a fluorescent dye label.
[Item 5] The probe according to any one of Items 1 to 4, wherein the label (B) is a label with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe.
[Item 6] The probe according to any one of Items 1 to 5, wherein the label (B) is a label with a fluorescence quenching dye that is quenched by interaction with guanine.
[Item 7] The probe according to any one of Items 1 to 6, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof.
[Item 8] The probe according to any one of Items 1 to 7, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).
[Item 9] The probe according to any one of Items 1 to 8, wherein in (B) above, the labeled terminal base is cytosine.
[Item 10] A method for detecting RS virus that may be contained in a specimen sample, using the probe according to any one of Items 1 to 9.
[Item 11] The following steps (1), (2), and (3):
(1) reverse transcribing target RNA in the specimen sample into cDNA;
(2) generating one or more nucleic acid amplification products using the cDNA of step (1) as a template; and (3) detecting the one or more nucleic acid amplification products of step (2) using one or more of the probes.
[Item 12] The method according to Item 11, wherein step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.
[Item 13] The method according to Item 12, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof.
[Item 14] The method according to any one of Items 11 to 13, wherein step (3) is carried out by melting curve analysis.
[Item 15] The method according to any one of Items 11 to 14, wherein the enzyme having reverse transcription activity used in the reverse transcription reaction of step (1) is a reverse transcriptase derived from M-MLV (Moloney Murine Leukemia Virus) or a mutant thereof.
[Item 16] The method according to any one of Items 10 to 15, wherein both subgroup A and B types of RS virus are detected.
[Item 17] The method according to any one of items 10 to 16, wherein RS virus is detected by distinguishing between subgroup A and subgroup B.
[Item 18] A reagent kit for detecting RS virus, comprising the probe according to any one of Items 1 to 9.

The present invention enables the simple, rapid, and highly sensitive detection of RS virus. Furthermore, the present invention also makes it possible to detect both type A and type B RS virus using a single probe. Furthermore, the present invention also makes it possible to distinguish and detect RS virus as either type A or type B. The use of the probe of the present invention, as well as detection methods, reagents, kits, etc., using the probe, enables the simple, rapid, and highly sensitive detection of RS virus, making a significant contribution to the field of clinical diagnosis.

This figure shows the correspondence between portions of the sequences (RSV A, RSV B) obtained by reverse transcribing the RNA genome sequences of RS virus subgroups A and B to DNA, and each probe used in the test examples. The double-underlined sequences indicate a region of 8 bases from positions 45 to 52 in the base sequence shown in SEQ ID NO: 1 or 2. The boxed portions in SEQ ID NOs: 3 to 7 and 10 to 14 indicate mismatched bases with the base sequence shown in SEQ ID NO: 1 or 2 or a base sequence complementary thereto. FIG. 1 shows a representative example of the results of Example 1 (a graph showing the detection results when melting curve analysis was performed using nucleic acid probe a. In the figure, RSV indicates the RS virus detection peak, and IC indicates the detection of the internal control). FIG. 1 shows a representative example of the results of Example 2 (a graph showing the detection results when melting curve analysis was performed using nucleic acid probe a. In the figure, RSV indicates the RS virus detection peak, and IC indicates the detection of the internal control).

The present invention will be described in more detail below by showing embodiments of the present invention, but the present invention is not limited to these.
All non-patent documents and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, the term "to" in this specification means "at least, at most," and for example, if the specification states "X to Y," it means "at least X and at most Y." In this specification, "and/or" means either one or both. In this specification, "comprise" encompasses the concepts of "consisting essentially of" and "consisting only of."

Furthermore, in this specification, nucleic acid primers may be simply referred to as primers, and nucleic acid probes and labeled probes may be simply referred to as probes, and these are collectively referred to as oligonucleotides.

In one embodiment, the present invention provides a method for detecting respiratory syncytial virus (RSV) using a labeled probe composed of a specific base sequence. This method enables simple, rapid, and highly sensitive detection of RSV. Furthermore, by designing a labeled probe that targets a specific region in the RSV genomic RNA, RSV subgroup A, which generally tends to become severe, can be reliably detected by melting curve analysis or other methods. Furthermore, a single labeled probe can detect both RSV subgroups A and B. In this specification, SEQ ID NOs: 1 and 2 represent base sequences corresponding to the DNA sequence obtained by reverse transcribing a portion of the RSV genomic RNA sequence (corresponding to the forward strand of the RSV genome sequence, which is a single-stranded negative-strand RNA virus). SEQ ID NO: 1 corresponds to the base sequence of RSV type A, and SEQ ID NO: 2 corresponds to the base sequence of RSV type B. The probe of the present invention is preferably a labeled probe (also referred to herein as a "nucleic acid probe") composed of a specific base sequence that targets the base sequence region set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[Method for detecting RS virus]
In one embodiment, the method for detecting RS virus that may be contained in a specimen sample is preferably a method using a nucleic acid probe described below. This method may be a method for determining the presence or absence of RS virus in a specimen sample. Furthermore, this method may be a method for quantifying RS virus contained in a specimen sample. In a specific embodiment, by using a single type of nucleic acid probe, RS virus that may be contained in a specimen sample, regardless of whether it is subgroup A or B, can be detected with high sensitivity, for example, in an RT-PCR-melting curve analysis method. In a further specific embodiment, RS virus that may be contained in a specimen sample can be distinguished and detected as either subgroup A or subgroup B, for example, based on the difference in detection temperature in a melting curve analysis method.

In a specific embodiment, a method for detecting RS virus that may be contained in a specimen sample includes at least the following steps (1), (2), and (3):
(1) reverse transcription (RT) of target RNA in a specimen sample into cDNA;
Preferably, the method includes: (2) generating one or more nucleic acid amplification products using the cDNA of step (1) as a template; and (3) detecting one or more nucleic acid amplification products of step (2) using one or more nucleic acid probes described below. Preferably, step (2) is performed by PCR reaction, and step (3) is performed by melting curve analysis (RT-PCR-melting curve analysis). Steps (1), (2), and (3) may be performed in the same reaction solution. Alternatively, two or more of steps (1), (2), and (3), for example, step (1) and step (2), may be performed consecutively or simultaneously.

[Step (1)]
In one embodiment, step (1) is preferably a step of generating cDNA from target RNA by performing a reverse transcription reaction using a nucleic acid primer and a reverse transcriptase. The reverse transcriptase is not particularly limited as long as it has reverse transcription activity, and examples include reverse transcriptases (RNA-dependent DNA polymerases) derived from M-MLV (Moloney Murine Leukemia Virus) and AMV (Avian Myeloblastosis Virus), as well as mutants thereof. Examples of such mutants include those in which one to three amino acids are deleted, substituted, inserted, and/or added from the wild-type amino acid sequence, and those exhibiting 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, examples include mutants lacking RNase H activity to increase the efficiency of cDNA synthesis. Furthermore, it is known that Tth DNA polymerase and its mutants also have reverse transcription activity under certain conditions, and can be used as the reverse transcriptase in the present invention.

The nucleic acid primer used in the reverse transcription reaction may also serve as one of the primers used in the nucleic acid amplification reaction in step (2). Furthermore, the conditions for the reverse transcription reaction are not particularly limited as long as the reaction proceeds. The temperature and time for the reverse transcription reaction (or the set temperature and time in the reverse transcription reaction apparatus) are, for example, preferably 37 to 55°C and 0 seconds to 60 minutes, more preferably 40 to 52°C and 1 to 30 minutes, and particularly preferably 42 to 50°C and 2 to 15 minutes.

[Step (2)]
In one embodiment, step (2) is preferably a step of generating a nucleic acid amplification product by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more nucleic acid primer sets). Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level at which it can be visualized, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene testing, and environmental testing. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably PCR (including RT-PCR), but is not limited to this.

(PCR reaction)
The PCR reaction is a reaction catalyzed primarily by DNA polymerase. PCR typically involves three steps: (i) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (ii) annealing of a primer to a template single-stranded DNA, and (iii) extension of the primer using a DNA polymerase, with this cycle being repeated. Examples of DNA polymerases include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In the present invention, it is preferable to use a DNA polymerase belonging to Family B because of its simplicity, speed, high sensitivity, and resistance to amplification inhibition by the sample. Furthermore, when step (3) is performed using melting curve analysis, it is preferable to use a DNA polymerase belonging to Family B that does not have 5'→3' exonuclease activity, also from the perspective of using a fluorescence quenching probe.

The conditions for the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be carried out at 80-100°C for approximately 0-300 seconds (e.g., approximately 0.5-300 seconds), and the second and subsequent (repeated) steps (i) may be carried out at 80-100°C for approximately 0.5-300 seconds, step (ii) may be carried out at 35-80°C for approximately 1-300 seconds, and step (iii) may be carried out at 35-85°C for approximately 1-300 seconds. It is preferable to repeat the cycle of steps (i) to (iii) 30-70 times. The temperature and time of the repeated cycles may be changed every 1-3 cycles.

(DNA polymerase)
The DNA polymerase that can be used in step (2) is preferably, but is not limited to, a DNA polymerase belonging to Family B. The Family B DNA polymerase is not particularly limited, but is preferably a DNA polymerase derived from Archea, more preferably a DNA polymerase derived from bacteria of the genera Pyrococcus and Thermococcus. Suitable DNA polymerases also include mutants of Family B archaea that do not lose their DNA polymerase activity. Examples of mutants include those with deletion, substitution, insertion, and/or addition of one to three amino acids in the wild-type amino acid sequence, or those exhibiting 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, DNA polymerase mutants include, but are not limited to, those intended for enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc.

DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity.

DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, as well as mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerase derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) are particularly suitable for use in the present invention due to their excellent extensibility and thermostability.

PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), and any of these can be used in the present invention.

(DNA polymerase derived from KOD)
As used herein, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to a DNA polymerase derived from Thermococcus kodakaraensis and its variants (e.g., a KOD-derived DNA polymerase in which 3' to 5' exonuclease activity has been deleted by substituting, deleting, inserting, and/or adding one to three amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, step (2) involves performing a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, a DNA polymerase belonging to Family A, KOD DNA polymerase has superior accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by sample-derived inhibitors. In the present invention, the use of such a KOD DNA polymerase is preferable in terms of simple, rapid, and highly sensitive detection of RS virus, as shown in the examples described below.

(Nucleic acid primer set)
The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify a nucleic acid fragment derived from RS virus with which a probe described below can form a complex. From the viewpoint of easily obtaining more sensitive determination results, the nucleic acid primer set is preferably a nucleic acid primer set that can amplify a nucleotide sequence that includes part or all of the nucleotide sequence of RS virus genomic RNA shown in SEQ ID NO: 1 or 2.

For example, the nucleic acid primer set preferably comprises a primer containing a base sequence of 20 to 35 consecutive bases in the region from bases 1 to 40 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence complementary to said base sequence, and a primer containing a base sequence of 20 to 35 consecutive bases in the region from bases 50 to 91 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence complementary to said base sequence, and one primer is complementary to the DNA extension product of the other primer.

More preferably, the primer set is composed of a primer containing a base sequence of 20 to 30 consecutive bases in the region from positions 1 to 31 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence complementary to said base sequence, and a primer containing a base sequence of 20 to 30 consecutive bases in the region from positions 60 to 91 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence complementary to said base sequence, wherein one primer is complementary to the DNA extension product of the other primer.

More preferably, the primer set is composed of a primer consisting of the base sequence shown in SEQ ID NO: 8 or a base sequence complementary to said base sequence, and a primer consisting of the base sequence shown in SEQ ID NO: 9 or a base sequence complementary to said base sequence, wherein one primer is complementary to the DNA extension product of the other primer.

The primer set may also include primers whose base sequences are such that 1 to 3 bases are substituted, deleted, inserted, or added in the base sequence of each primer.

[Step (3)]
The embodiment of step (3) is not particularly limited and can be performed by any method known in the art. RS viruses to be detected are prone to mutation, and various subtypes of genome sequences exist. Here, mismatches between the base sequence of a primer or probe and the base sequence of a target RS virus mutant strain can reduce the binding strength of the primer or probe to viral RNA or the nucleic acid amplification product derived therefrom (target nucleic acid). In particular, if there are many mismatches between the target nucleic acid and the probe in real-time RT-PCR, the probe cannot sufficiently bind to the target nucleic acid, resulting in a delayed or complete absence of an apparent rise in the amplification curve, which can lead to false negatives. In the case of melting curve analysis, step (3) is performed after RT-PCR is completed. Therefore, even if there are mismatches in the primer or probe, detection is possible as long as the final nucleic acid amplification product is obtained. Therefore, the impact of mismatches can be reduced compared to real-time RT-PCR. Therefore, it is particularly preferable to detect the nucleic acid amplification product using melting curve analysis in step (3). Furthermore, by detecting nucleic acid amplification products using melting curve analysis, it may be possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the reverse transcription reaction to the completion of melting curve analysis).

In one embodiment, step (3) comprises the following steps (3-1) and (3-2):
The method preferably includes the steps of: (3-1) hybridizing the nucleic acid amplification product of step (2) with a nucleic acid probe to form a complex; and (3-2) detecting the complex of step (3-1). In order to obtain highly sensitive results in detecting RS virus, it is preferable to use a nucleic acid probe described below that can specifically react with the RS virus-derived nucleic acid amplification product of step (2) to form a complex.
The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product with the nucleic acid probe. Examples of such temperature conditions include, but are not limited to, a temperature that is at least 5°C lower, more preferably at least 10°C lower than the Tm value of the nucleic acid probe.

(Nucleic acid probe)
The nucleic acid probe of the present invention may be a probe for detecting RS virus, which has at least the following characteristics (A) and (B):
(A) containing the following base sequence (A-1) or (A-2):
(A-1) A base sequence of at least 10 consecutive bases in a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which base sequence includes at least a base sequence complementary to the 45th to 52nd bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or (A-2) A base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence of (A-1); and (B) Only either the 5' end or the 3' end is labeled.
By using a nucleic acid probe having such characteristics, RS virus can be detected with high sensitivity, for example, even in melting curve analysis.

The nucleic acid probe of the present invention is not particularly limited as long as it has the base sequence (A). However, when the nucleic acid probe is labeled with a fluorescent quenching dye that quenches fluorescence through interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

In one embodiment, the nucleic acid probe of the present invention can detect RS viruses (including mutant strains of RS viruses) whose genome contains a base sequence that is 85% or more identical to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and is preferably a nucleic acid probe that can detect RS viruses whose genome contains a base sequence that is 90% or more identical to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more.

In a specific embodiment, the nucleic acid probe of the present invention may be a probe containing a base sequence (A-2) in which one to three bases have been substituted, deleted, inserted, or added in the base sequence shown in (A-1). The number of substituted, deleted, inserted, or added bases may preferably be one or two. When a probe contains such a base substitution, deletion, insertion, or addition, it is said to contain mismatched bases, and probes containing such mismatched bases can also be preferably used in the present invention. The use of such probes containing mismatched bases has the advantage of making it easier to detect both RS virus subgroups A and B.

As used herein, "containing a mismatched base" (or simply "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (if the target nucleic acid becomes double-stranded after the nucleic acid amplification reaction, the base sequence of one of the single-stranded nucleic acids resulting from the dissociation of the double strand). For example, if a cytosine base is present in the base sequence of the target nucleic acid, this means that the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., adenine, cytosine, thymine, or universal base). For example, when detecting a region of a target nucleic acid that is susceptible to mutation, a mismatched base (e.g., a universal base) can be selected for the position of the probe that corresponds to the susceptible base.

When the nucleic acid probe of the present invention contains a mismatched base, the position of the mismatched base is not particularly limited as long as it does not inhibit the effects of the present invention. From the perspective of more reliably detecting nucleic acid amplification products, it is preferable that the mismatched base is not a terminal base of each probe. For example, the position of the mismatched base is preferably within 8 mers before and after the center of the total length n of the base sequence constituting the probe ((n+1)/2 when n is odd, n/2 when n is even), more preferably within 7 mers before and after, even more preferably within 6 mers before and after, and even more preferably within 5 mers before and after.

In one preferred embodiment, the nucleic acid probe of the present invention is a probe comprising a base sequence of at least 10 or more consecutive bases in a base sequence complementary to the base sequence shown at positions 27 to 61 of the base sequence shown in SEQ ID NO: 1 or 2, and including at least a base sequence complementary to positions 45 to 52 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence. Preferably, the nucleic acid probe is a probe comprising a base sequence of at least 10 or more consecutive bases in a base sequence complementary to the base sequence shown at positions 40 to 61 of the base sequence shown in SEQ ID NO: 1 or 2, and including at least a base sequence complementary to positions 45 to 52 of the base sequence shown in SEQ ID NO: 1 or 2, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence. In these probes, the number of substituted, deleted, inserted or added bases in the base sequence complementary to positions 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is preferably 0, 1, or 2, and more preferably 0 or 1. By reacting such a probe with the nucleic acid amplification product, RS virus can be more effectively detected.

The length of the nucleic acid probe of the present invention is not particularly limited, but is, for example, 10 bases or more, preferably 14 bases or more, and typically 26 bases or less, preferably 20 bases or less. The probe length is more preferably 14 to 26 bases, and even more preferably 14 to 20 bases. Using a probe of such a length enables RS virus detection with higher sensitivity.

In a specific preferred embodiment, specific examples of nucleic acid probes of the present invention include probes containing a base sequence complementary to any of the base sequences set forth in SEQ ID NOS: 3 to 7, or a base sequence in which one to three bases have been substituted, deleted, inserted, or added within such a base sequence. By using a probe containing such a specific base sequence, RS virus can be detected with even greater sensitivity.

The above-mentioned probe is characterized in that only either the 5' end or the 3' end is labeled. In one embodiment, the nucleic acid probe of the present invention is preferably labeled to produce quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity to a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably labeled to produce quenching. There are no particular limitations on the labeling substance, but a fluorescent dye is more preferred.

The fluorescent dye may be either a fluorescent substance that emits fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product. Particularly preferred are fluorescent quenching dyes that quench fluorescence by interacting with guanine when hybridized with a target nucleic acid amplification product (e.g., fluorescent quenching dyes that quench fluorescence by interacting with guanine). Specific examples include fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODI Examples of such quenching dyes include, but are not limited to, at least one fluorescence quenching dye selected from the group consisting of BODIPY-FL/C3, BODIPY-FL/C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, and BODIPY-665.

More specifically, examples of fluorescence quenching dyes that quench through interaction with guanine include at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

In certain preferred embodiments, probes labeled with a fluorescence quenching dye and having a terminal base of cytosine are more preferred. When such probes hybridize to nucleic acid amplification products, they form base pairs with guanine bases in the nucleic acid amplification products, thereby quenching the fluorescence, making it very easy to measure changes in the fluorescence intensity of the reaction solution.

When the probe hybridizes, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5,354,216, and this technology can also be referenced in the present invention. In other words, when the probe hybridizes, fluorescence can be quenched if the guanine base in the nucleic acid amplification product is located within a range of, for example, 1 to 3 bases of the cytosine base of the probe (a base that forms a base pair with a cytosine base is counted as 1).

Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5,354,216, and this technology can also be used as a reference for the present invention. For example, when the probe hybridizes, quenching can be achieved if a guanine base in the nucleic acid amplification product is located within a range of, for example, 1 to 3 bases of the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

In a particularly preferred embodiment, the nucleic acid probe of the present invention is used in step (3). Using the nucleic acid probe of the present invention in this manner makes it possible to detect RS virus subgroups A and/or B. The method of the present invention is particularly effective in detecting RS virus subgroup A, which is generally considered to be more likely to develop severe symptoms. Furthermore, the method of the present invention may use one type of probe of the present invention, or a combination of two or more types. In a particularly preferred embodiment, the method of the present invention can detect both RS virus subgroups A and B using only one type of labeled probe.

In one embodiment, step (3) may include at least one of the following steps (3-a), (3-b), and (3-c):
(3-a) Simultaneously with step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) in step (2) in real time.
(3-b) After completion of step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution to monitor the progress of the reaction (nucleic acid amplification reaction) in step (2) at an endpoint.
(3-c) After the step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution.
Step (3-a), (3-b), or (3-c) allows for simple, rapid, and highly sensitive detection of the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe. Steps (3-a), (3-b), and (3-c) may be performed in combination; for example, both steps (3-a) and (3-b), or both steps (3-a) and (3-c) may be performed. In one embodiment, step (3-b) or (3-c) is preferred from the viewpoint of more rapid detection of a nucleic acid amplification product. Step (3-c), i.e., melting curve analysis, is particularly preferred.

(Step (3-a))
Step (3-a) is a method for monitoring the progress of the nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

(Step (3-b))
In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the endpoint, thereby enabling rapid detection of the target nucleic acid contained in the specimen. Furthermore, the approximate amount of the target nucleic acid can be estimated by comparing the fluorescence intensity at the endpoint.
For example, the progress of a nucleic acid amplification reaction is monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescence quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured and compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming whether or not the target nucleic acid has been amplified. Alternatively, the presence or absence of the target nucleic acid in a test sample can be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. A control reaction solution is a reaction solution to which a sample known to be negative or positive has been added instead of the test sample to be measured.
The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and simple detection, it is preferable to measure at the end point.

(Step (3-c))
In step (3-c), measuring the temperature dependence of fluorescence intensity can specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be determined by first differentiating the obtained fluorescence intensity with respect to temperature. Furthermore, the fluorescence intensity may be converted into a fluorescence quenching rate or the like depending on the purpose.
The detection, analysis, etc. of a target nucleic acid using the Tm value is called melting curve analysis. Generally, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that does not form a double strand and remains single-stranded. Since the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms in a target nucleic acid. The base sequence polymorphism referred to here includes single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

As an example, melting curve analysis is also applied to SNP analysis. If there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases will mismatch when the probe hybridizes, and the Tm value will generally be low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm values.

[Specimen sample]
The specimen sample that can be used in the present invention is not particularly limited as long as it may contain RS virus. Examples include, but are not limited to, oral scrapings, throat swabs, nasopharyngeal swabs, nasal swabs, nasal aspirates, sputum, bronchial lavage fluid, alveolar lavage fluid, and saliva collected from subjects suspected of RS virus infection. When a biological sample is used as the specimen sample to be measured, pretreatment such as dilution, suspension, centrifugation, or enzyme treatment, or nucleic acid extraction may be performed depending on the biological sample, although this is not particularly limited.

There are no particular limitations on the collection and preparation methods for the specimen sample, and known methods can be used depending on the type and purpose of the specimen sample. In certain preferred embodiments, the specimen sample does not need to be a sample from which RNA has been isolated and purified. For example, a specimen collected from a living organism may be subjected to proteolytic denaturation treatment using a protease (e.g., proteinase K) and/or heat treatment (e.g., at 60-100°C for 1 second to 10 minutes) to decompose and denaturate RNase (ribonuclease) and DNase (DNA-degrading enzyme) present in the specimen, and the resulting sample may be used as is.

The method for nucleic acid extraction is not particularly limited, and known methods can be used depending on the type of specimen and purpose. Nucleic acid extraction may involve extracting primarily RNA, or may involve extracting nucleic acids without distinguishing between RNA and DNA. For nucleic acid extraction, kits available from various manufacturers may be used, such as the QIAamp Viral RNA Mini Kit (QIAGEN). An automated extraction and purification system may also be used.

In certain preferred embodiments, the specimen sample may be one that has been prepared without the nucleic acid purification step that is typically considered essential in conventional nucleic acid amplification reactions. Nucleic acid purification requires specialized reagents and is problematic in that the process is cumbersome, time-consuming, and labor-intensive. Using a specimen sample that has been prepared without the nucleic acid purification step can shorten the time from specimen collection to obtaining genetic test results. For example, the time from specimen collection to obtaining genetic test results can be reduced to within one day, preferably within half a day, more preferably within six hours, even more preferably within three hours, and especially preferably within two hours (e.g., within one hour). Thus, when the method of the present invention is performed using a specimen sample that has not undergone a nucleic acid purification step, the effort of nucleic acid purification can be eliminated, enabling RS virus to be detected easily and quickly.

[Reagents for detecting RS virus]
In another embodiment, the present invention provides a reagent for detecting RS virus. The reagent preferably contains at least the components necessary for the reverse transcription reaction, nucleic acid amplification reaction, and detection, in addition to the nucleic acid probe of the present invention described above. These necessary components can be any known components. For example, the reagent of the present invention preferably contains at least a nucleic acid primer for reverse transcription, a nucleic acid primer set for PCR, a reverse transcriptase, a DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and an inorganic salt such as a magnesium salt. Multiple sets of nucleic acid primer sets for PCR that also serve as nucleic acid primers for reverse transcription and nucleic acid probes for detection can be included to amplify multiple regions of RS virus. The concentration of each component can be adjusted as appropriate; for example, the nucleic acid probe is preferably 0.01 to 1 μM, and more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, it is preferable that each nucleic acid probe contained in the probe set be within the above-mentioned concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. The reverse transcriptase is preferably 0.01 to 10 U/μL, more preferably 0.02 to 2 U/μL. The DNA polymerase is preferably 0.01 to 1 U/μL, more preferably 0.02 to 0.5 U/μL. The deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

Furthermore, the reagents of the present invention may contain additives known in the art for the purposes of suppressing nonspecific amplification or promoting the reaction. Examples of additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies and phosphate. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, and Nonidet P40. Furthermore, in order to reduce degradation of the target RS virus genomic RNA, the reagent of the present invention may contain an inhibitor or suppressor of RNase (e.g., an RNase inhibitor). Furthermore, in order to facilitate the determination of false negatives, the reagent of the present invention preferably also contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more types.

[Reagent kit for detecting RS virus]
In another embodiment, the present invention provides a reagent kit for detecting RS virus. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe or reagent of the present invention described above and is configured to detect (including differentiate) RS virus. For example, the kit of the present invention can optionally contain a reagent capable of detecting or quantifying the presence of the target substance and/or instructions for use, etc. For example, the kit of the present invention can contain the nucleic acid probe, components necessary for the reverse transcription reaction, components necessary for the nucleic acid amplification reaction, and components necessary for detecting the amplification product, all sealed in the same container or in separate containers, packaged in a single package and provided with information on how to use the kit. The kit of the present invention can also contain a positive control solution and/or a negative control solution.

The present invention will be explained in detail below based on examples. The present invention is not limited to the following examples.

Test Example 1: Evaluation of Nucleic Acid Probe 1
(1) Sample Preparation Crudely extracted RNA from subgroup A of RS virus was serially diluted with sterile water to prepare a sample with 100 copies per test.
(2) Reverse transcription, nucleic acid amplification, melting curve analysis, and judgment The above samples were added to the following reagents, and RS virus subgroup A was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis. A positive result was judged when a peak was obtained in the melting curve analysis.

(reagent)
To compare the performance of each of the labeled probes a to j designed as shown in Figure 1, the following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were adjusted according to the instructions in their respective instruction manuals (ReverTra Ace (registered trademark) was used at 0.1 U/μL). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally.
3.0 μM primer shown in SEQ ID NO: 8 0.5 μM primer consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 9 0.4 μM each of probes a to j consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 3 to 7, 13, and 14, or the base sequences shown in SEQ ID NOs: 10 to 12 (all labeled at the 3' end with CR6G)

(Reverse transcription, nucleic acid amplification and melting curve analysis)
42°C for 2 minutes, 97°C for 15 seconds (one cycle)
97℃ 1 second 58℃ 3 seconds 63℃ 5 seconds (50 cycles)
94°C for 30 seconds, 39°C for 30 seconds, 40°C to 75°C (temperature rises at 0.09°C/second)
The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

(3) Results Figure 2 is a detection graph obtained when RS virus subgroup A RNA was detected by nucleic acid amplification and melting curve analysis using probe a consisting of a base sequence complementary to SEQ ID NO: 3. As shown in Figure 2, probe a gave a detection peak at around 49°C.

The measurement results using the nucleic acid probes used in Test Example 1 are summarized in Table 1. By selecting nucleic acid probes (probes a-e) containing a base sequence complementary to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, it was possible to detect RS virus subgroup A RNA. However, completely unexpectedly, when similar nucleic acid probes were designed with the normal strand base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, even if they contained a region corresponding to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, they were not successfully detected by melting curve analysis (probes f-h). Furthermore, it was revealed that even when nucleic acid probes were designed with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, they were unable to detect RNA if they did not contain a region corresponding to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (probes i-j).

Furthermore, as shown in Figure 1, probes a, c, d, and e are not completely identical to the base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and contain mismatched bases, but they were shown to be capable of detecting RS virus subgroup A with sufficient sensitivity.

Test Example 2: Evaluation of Nucleic Acid Probes 2
(1) Sample Preparation Crudely extracted RNA from subgroup B of RS virus was serially diluted with sterile water to prepare a sample with 100 copies per test.
(2) Reverse transcription, nucleic acid amplification, melting curve analysis, and determination In the same manner as in Test Example 1, detection of RS virus subgroup B was carried out.

(reagent)
The following solutions were prepared using each of the probes a, c, d, f, and i used in Test Example 1, GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). As in Test Example 1, GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were used in the amounts adjusted according to the instructions in their respective instruction manuals (ReverTra Ace (registered trademark) was used at 0.1 U/μL). An internal control (IC) of a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally.
3.0 μM Primer shown in SEQ ID NO: 8 0.5 μM Primer consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 9 0.4 μM Each of probes a, c, d, f, and i (all labeled at the 3' end with CR6G)

(Reverse transcription, nucleic acid amplification and melting curve analysis)
The test was carried out under the same conditions as in Test Example 1.

(3) Results Figure 3 shows a detection graph obtained when RS virus subgroup B RNA was detected by nucleic acid amplification and melting curve analysis using probe a consisting of a base sequence complementary to SEQ ID NO: 3. As shown in Figure 3, probe a gave a detection peak at around 54°C.

The measurement results using the nucleic acid probe used in Test Example 2 are summarized in Table 2. It was demonstrated that RS virus subgroup B RNA can also be detected by selecting and using nucleic acid probes (probes a, c, and d) containing a base sequence complementary to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, as in Test Example 1, it was demonstrated that subgroup B RNA could not be detected when a similar nucleic acid probe was designed with the base sequence of the normal strand shown in SEQ ID NO: 1 or 2 (probe f) even though it contained a region corresponding to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or when a nucleic acid probe was designed with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but did not contain a region corresponding to the 8 bases 45 to 52 of the base sequence shown in SEQ ID NO: 1 or 2 (probe i).

Furthermore, compared to the results of Test Example 1, the detection temperatures for RS virus subgroup A and subgroup B using the probes of the present invention (probes a, c, and d) were different. Therefore, by utilizing this difference in detection temperature, it was found that the probes of the present invention can also distinguish between RS virus subgroups A and B.

By using the nucleic acid probe of the present invention, RS virus that may be present in a sample can be detected simply, quickly, and with high sensitivity, for example, by melting curve analysis. The present invention makes it possible to detect RS virus with high reliability, making a significant contribution to clinical diagnosis, etc.

Claims (17)

A probe for detecting RS virus, having the following characteristics (A) and (B):
(A) A base sequence of (A-1) or (A-2) below:
(A-1) a base sequence of 14 to 26 consecutive bases in a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which base sequence includes at least a base sequence complementary to the 45th to 52nd bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or (A-2) a base sequence in which one base has been substituted, deleted, inserted or added in the base sequence of (A-1); and (B) only either the 5' end or the 3' end is labeled. The probe according to claim 1 , wherein the base sequence of (A) comprises a base sequence complementary to the base sequence shown in any one of SEQ ID NOs: 3 to 7. The probe according to claim 1 or 2 , wherein the label (B) is a fluorescent dye label. The probe according to any one of claims 1 to 3, wherein the label (B) is a label with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe. The probe according to any one of claims 1 to 4 , wherein the label (B) is a label with a fluorescence quenching dye that is quenched by interaction with guanine. The probe according to any one of claims 1 to 5 , wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives. The probe according to any one of claims 1 to 6, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran- 3- carboxylic acid (Pacific Blue). The probe according to any one of claims 1 to 7 , wherein in (B), the labeled terminal base is cytosine. A method for detecting RS virus that may be contained in a specimen sample, using the probe according to any one of claims 1 to 8 . The following steps (1), (2), and (3):
(1) reverse transcribing target RNA in the specimen sample into cDNA;
10. The method of claim 9, comprising: (2) generating one or more nucleic acid amplification products using the cDNA of step (1) as a template; and (3) detecting the one or more nucleic acid amplification products of step ( 2 ) using one or more of the probes. 11. The method according to claim 10 , wherein step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. The method according to claim 11 , wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof. The method according to any one of claims 10 to 12 , wherein step (3) is carried out by melting curve analysis. The method according to any one of claims 10 to 13 , wherein the enzyme having reverse transcription activity used in the reverse transcription reaction in step (1) is a reverse transcriptase derived from M-MLV (Moloney Murine Leukemia Virus) or a mutant thereof. The method according to any one of claims 9 to 14 , wherein both subgroups A and B of RS virus are detected. The method according to any one of claims 9 to 15 , wherein RS virus is detected by distinguishing between subgroup A and subgroup B. A reagent kit for detecting RS virus, comprising the probe according to any one of claims 1 to 8 .