JP7589458B2 - Oligonucleotides for use in detecting coronavirus (SARS-CoV-2) and detection method thereof - Google Patents

Description

The present invention relates to an oligonucleotide for rapid, highly sensitive and specific detection of the 2019 novel coronavirus (hereinafter referred to as SARS-CoV-2) contained in a sample, and a method for detecting SARS-CoV-2 using the oligonucleotide.

The novel coronavirus disease (hereafter referred to as COVID-19) is caused by the SARS-CoV-2 virus and was confirmed in Wuhan, Hubei Province, People's Republic of China in 2019. The World Health Organization (WHO) declared COVID-19 a "Public Health Emergency of International Concern (PHEIC)." Subsequently, on March 11, 2020, based on the global spread of infection and the severity of the disease, it announced that COVID-19 could be considered a pandemic (global epidemic).

The Coronaviridae family is a positive-sense, single-stranded, enveloped virus measuring 80-160 nm in diameter that infects a variety of animals, including humans, dogs, pigs, cattle, and camels. Based on genetic characteristics, they are classified into genera α, β, γ, and δ. Viruses that mainly cause cold symptoms in humans include the 229E and NL63 strains of the αcoronavirus genus, and the OC43 and HKU1 strains of the βcoronavirus genus. These four types of viruses account for 10-15% of colds (35% during epidemics). In addition to these, SARS (severe acute respiratory syndrome) coronavirus was identified in 2003, and MERS (middle east respiratory syndrome) coronavirus was identified in 2012. Both of these coronaviruses cause severe infections, mainly pneumonia, in humans, with extremely high mortality rates of approximately 10% for the former and over 30% for the latter (Non-Patent Document 1). SARS-CoV-2 belongs to the same β-coronavirus family as SARS and MERS. Based on complete base analysis and phylogenetic tree analysis, SARS-CoV-2 has been found to share 75-80% homology with SARS coronavirus and 85-88% homology with bat coronaviruses. For this reason, SARS-CoV-2 is believed to have originated from a bat virus.

Initial symptoms are mild and include fever, fatigue, dry cough, loss of appetite, muscle pain, difficulty breathing, phlegm, and sore throat. In some cases, the loss of smell and taste may occur, and in severe cases, pneumonia may develop. Initial symptoms usually last for about 5 to 7 days and will gradually improve if they do not become severe.

When the condition worsens, breathing becomes difficult. In addition to pneumonia, inflammation of other respiratory organs, such as upper respiratory tract infections and bronchitis, may also be observed. Even if severe pneumonia occurs, it will gradually recover with treatment for the symptoms, but if the condition worsens and becomes serious, it can lead to acute respiratory syndrome (ARDS), septic shock, multiple organ failure, and in some cases death.

With normal viral infections, the likelihood of infecting others is highest when symptoms are most pronounced, but it is believed that COVID-19 patients can be infected when they are asymptomatic, when symptoms are mild, and when symptoms are severe. In particular, identifying asymptomatic pathogen carriers who are infected with the virus but have no symptoms and are capable of infecting others is of utmost importance in preventing the spread of infection and in-hospital infections. Furthermore, large-scale screening tests are considered effective in identifying asymptomatic individuals, and a test method with sufficient processing capacity and high sensitivity is required.

Highly sensitive testing methods include genetic testing methods that amplify and detect viral nucleic acids. However, the process of extracting viral nucleic acids from samples is complicated, and a certain level of training is required from the person carrying out the work to obtain reliable results. In addition, it takes time to obtain results, and the method lacks speed. On the other hand, antigen-antibody testing methods are characterized by their simplicity, high processing capacity, and high speed, but their sensitivity is not as high as that of genetic testing, and there is a possibility of false negatives.

In the TRC method used in the present invention (Patent Document 2, Patent Document 3), an integrated device from purification to detection is commercially available, and results can be obtained in about one hour, making it sufficiently rapid. In addition, the sensitivity and specificity are comparable to other detection methods that use nucleic acid amplification.

Because the nucleic acid to be amplified for the new coronavirus (SARS-CoV-2) has a high base sequence homology with other coronaviruses, it has been extremely difficult to design primer sets and oligonucleotide probes that can detect the gene with high sensitivity and specificity. It has been particularly difficult to distinguish it from the coronavirus (SARS-CoV) that was prevalent in 2003. When using an amplification method that can amplify RNA under relatively low constant temperatures (e.g., 40°C to 50°C), the nucleic acid to be amplified is more likely to form higher-order structures, making it even more difficult to design the primer sets and oligonucleotide probes.

JP 2009-131174 A JP 2000-14400 A JP 2001-37500 A

Wang C, et al: A novel coronavirus outbreak of global health concern. Lancet 395: 470-473, 2020. doi: 10.1016/S0140-6736(20)30185-9.

The object of the present invention is to provide a specific oligonucleotide that amplifies nucleic acid derived from the novel coronavirus (SARS-CoV-2) present in a sample with high sensitivity and rapidity while reducing the occurrence of false positives, and a method for detecting the novel coronavirus (SARS-CoV-2) using the oligonucleotide.

The inventors have conducted extensive research to solve the above problems, and as a result have completed the present invention. That is, the present invention is as follows.

(1) An oligonucleotide for detecting the 2019 novel coronavirus (SARS-CoV-2), characterized in that it consists of a sequence of 10 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 1 or its complementary sequence.
(2) The oligonucleotide described in claim 1, characterized in that the oligonucleotide (1) is labeled with a fluorescent dye and is configured so that its fluorescent properties change when it forms a complementary double-stranded chain.
(3) The oligonucleotide according to claim 2, characterized in that the oligonucleotide (2) is labeled with an intercalative fluorescent dye.

(4) A method for detecting the 2019 novel coronavirus (SARS-CoV-2), comprising using the oligonucleotide according to (1).
(5) The detection method according to (4), characterized in that the oligonucleotide according to (2) is used.
(6) The detection method according to (5), wherein the oligonucleotide is labeled with an intercalative fluorescent dye.
(7) The detection method according to any one of (4) to (6), characterized in that a pair of primer sets is used.
(8) The detection method described in (7), characterized in that the primer set comprises a first primer consisting of a sequence of 10 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 6 or its complementary sequence, and a second primer consisting of a sequence of 10 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 13 or its complementary sequence.
(9) The detection method according to (8), characterized in that the first primer consists of 15 to 30 consecutive bases in the base sequence set forth in SEQ ID NO: 6 or its complementary sequence, and the second primer consists of 15 to 30 consecutive bases in the base sequence set forth in SEQ ID NO: 13 or its complementary sequence.

The oligonucleotides of the present invention can specifically detect the base sequence of the RNA of the novel coronavirus (SARS-CoV-2) or its complementary sequence by hybridizing to a portion of the sequence specific to the RNA of the novel coronavirus (SARS-CoV-2) or its complementary sequence.

The oligonucleotide of the present invention and the method for detecting the novel coronavirus (SARS-CoV-2) using the oligonucleotide can rapidly and sensitively detect the novel coronavirus (SARS-CoV-2) contained in a sample, and is highly specific with extremely few false positives, i.e., it does not detect other coronavirus species. This makes it possible to promptly present the test results to a doctor, which is believed to contribute to preventing the spread of infection and early improvement of symptoms through the administration of appropriate medication.

The present invention is described in detail below.

In the present invention, samples include nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid, tracheal aspirates, pleural effusion, blood, urine, and stool.

The detection of a nucleic acid containing the nucleic acid base sequence of the RNA of the novel coronavirus (SARS-CoV-2) or its complementary sequence using the oligonucleotide of the present invention can be performed using a conventionally known nucleic acid detection method.
(A) A method using electrophoresis or liquid chromatography,
(B) Hybridization methods using oligonucleotide probes labeled with a detectable label;
(C) a method using a fluorescent dye-labeled oligonucleotide designed to change its fluorescence characteristics by hybridizing with a part of the base sequence of an amplification product obtained by amplifying a nucleic acid containing the specific base sequence or its complementary sequence using a set of primers;
Examples of the fluorescent dye-labeled oligonucleotide (C) include a fluorescent dye-labeled oligonucleotide that utilizes FRET (fluorescence resonance energy transfer) and an oligonucleotide labeled with an intercalating fluorescent dye.

An example of an oligonucleotide labeled with the intercalating fluorescent dye is an oligonucleotide that is specifically hybridizable with a portion of a nucleic acid containing the base sequence of the RNA of the novel coronavirus (SARS-CoV-2) or its complementary sequence, and that is labeled with an intercalating fluorescent dye via a suitable linker at the 3' end, 5' end, phosphate diester portion, or base portion of the oligonucleotide. When the oligonucleotide forms a complementary double strand with the base sequence (or its complementary sequence) of the RNA of the novel coronavirus (SARS-CoV-2), the intercalating fluorescent dye portion intercalates into the complementary double strand portion, changing the fluorescent properties. There is no particular limitation on the intercalating fluorescent dye to be used for labeling, and it may be appropriately selected from commonly used fluorescent dyes such as oxazole yellow, thiazole orange, ethidium bromide, and hemicyanine, and their derivatives, taking into consideration the fluorescent intensity and fluorescent properties. Except when labeling the 3' end with a fluorescent dye, the 3' end of the oligonucleotide should be appropriately modified with glycolic acid or the like to prevent nucleic acid elongation reaction from that end.

An example of an oligonucleotide constituting the oligonucleotide labeled with the intercalating fluorescent dye is an oligonucleotide that can specifically hybridize with a nucleic acid containing a base sequence of RNA possessed by the novel coronavirus (SARS-CoV-2) or its complementary sequence, and contains a consecutive base sequence of at least 10 bases in the base sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof. More preferably, the oligonucleotide can hybridize to the base sequence described in SEQ ID NO: 2 (the base sequence from 29212 to 29228 in GenBank No. MN908947.1) or its complementary sequence, the oligonucleotide can hybridize to the base sequence described in SEQ ID NO: 3 (the base sequence from 29216 to 29235 in GenBank No. MN908947.1) or its complementary sequence, the oligonucleotide can hybridize to the base sequence described in SEQ ID NO: 4 (the base sequence from 29228 to 29245 in GenBank No. MN908947.1) or its complementary sequence, and the oligonucleotide can hybridize to the base sequence described in SEQ ID NO: 5 (the base sequence from 29239 to 29258 in GenBank No. MN908947.1) or its complementary sequence.

Examples of hybridization conditions for the oligonucleotide and its complementary sequence in the present invention include conditions at 42°C in the presence of 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate, and nucleic acid amplification conditions described in the Examples of this specification. In addition, as long as the base sequences of the first and second primers can hybridize with the base sequence of the RNA of the novel coronavirus (SARS-CoV-2) sufficiently specifically and efficiently under the conditions, the base sequences of the first and second primers may have substitutions, deletions, additions, or modifications compared to the specific base sequence. The length of the first and second primers can be set arbitrarily, but is preferably in the range of 10 to 50 bases.

The present invention is characterized by using an oligonucleotide capable of specifically hybridizing to the base sequence described in SEQ ID NO: 6 (the base sequence from bases 29135 to 29206 in GenBank No. MN908947.1) as a first primer having a sequence complementary to the 5'-end of the base sequence of RNA of the novel coronavirus (SARS-CoV-2) contained in the sample, and an oligonucleotide capable of specifically hybridizing to the base sequence described in SEQ ID NO: 13 (the base sequence from bases 29280 to 29396 in GenBank No. MN908947.1) as a second primer having a sequence homologous to the 3'-end of the base sequence of RNA of the novel coronavirus (SARS-CoV-2) contained in the sample.

Of these, it is preferable that the first primer and the second primer consist of 15 to 30 bases.

An example of the first primer is an oligonucleotide having 15 to 30 consecutive bases in the base sequence set forth in SEQ ID NO: 6 or its complementary sequence. More specific examples include SEQ ID NO: 7 (complementary sequence of the base sequence from 29135 to 29154 of GenBank No. MN908947.1), SEQ ID NO: 8 (complementary sequence of the base sequence from 29142 to 29161 of GenBank No. MN908947.1), SEQ ID NO: 9 (complementary sequence of the base sequence from 29155 to 29174 of GenBank No. MN908947.1), SEQ ID NO: 10 (complementary sequence of the base sequence from 29166 to 29185 of GenBank No. MN908947.1), SEQ ID NO: 11 (complementary sequence of the base sequence from 29166 to 29185 of GenBank No. MN908947.1), and SEQ ID NO: 12 (complementary sequence of the base sequence from 29166 to 29185 of GenBank No. MN908947.1). Examples of such oligonucleotides include oligonucleotides consisting of the base sequence set forth in SEQ ID NO: 12 (the complementary sequence of the base sequence from 29179 to 29196 of GenBank No. MN908947.1) and SEQ ID NO: 12 (the complementary sequence of the base sequence from 29187 to 29206 of GenBank No. MN908947.1).

An example of the second primer is an oligonucleotide consisting of 15 to 30 consecutive bases in the base sequence set forth in SEQ ID NO: 13 or its complementary sequence. More specific examples include SEQ ID NO: 14 (the base sequence from 29280 to 29299 in GenBank No. MN908947.1), SEQ ID NO: 15 (the base sequence from 29295 to 29310 in GenBank No. MN908947.1), SEQ ID NO: 16 (the base sequence from 29307 to 29326 in GenBank No. MN908947.1), SEQ ID NO: 17 (the base sequence from 29324 to 29342 in GenBank No. MN908947.1), SEQ ID NO: 18 (the base sequence from 29325 to 29342 in GenBank No. MN908947.1), and SEQ ID NO: 19 (the base sequence from 29326 to 29342 in GenBank No. MN908947.1). Examples of such oligonucleotides include oligonucleotides having the base sequences set forth in SEQ ID NO: 19 (the base sequence from 29340 to 29359 of GenBank No. MN908947.1), SEQ ID NO: 20 (the base sequence from 29365 to 29387 of GenBank No. MN908947.1), and SEQ ID NO: 21 (the base sequence from 29373 to 29396 of GenBank No. MN908947.1).

Among these, a primer set in which the first primer is an oligonucleotide consisting of a base sequence set forth in any one of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, and the second primer is an oligonucleotide consisting of a base sequence set forth in any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, and 21, is preferred, in terms of being capable of rapidly and specifically detecting RNA possessed by the novel coronavirus (SARS-CoV-2). Among these, a primer set in which the combination of the first primer and the second primer is an oligonucleotide consisting of a base sequence set forth in any one of SEQ ID NOs: 8 and 20, 10 and 20, 10 and 14, 10 and 17, 10 and 19, and 10 and 21 is even more preferred.

The primer set of the present invention is useful as a primer set for amplifying a nucleic acid containing the base sequence of RNA possessed by the novel coronavirus (SARS-CoV-2) or its complementary sequence, using a nucleic acid amplification method commonly used by those skilled in the art, such as RT-PCR. Furthermore, when a promoter for RNA polymerase is further added to the 5'-end of either the first or second primer, a nucleic acid containing a base sequence to which a promoter for RNA polymerase has been added or its complementary sequence is synthesized using an RNA polymerase corresponding to the promoter, and therefore, this is preferable in that RNA can be amplified from these nucleic acids using a method for amplifying RNA at a constant temperature, such as the Nucleic Acid Sequence Based Amplification (NASBA) method, the Transcription-Mediated Amplification (TMA) method, or the Transcription-Reverse Transcription Concerted reaction (TRC) method. The promoter added to the 5' end of the primer may be a promoter compatible with the RNA polymerase used in RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, which are commonly used in the field of molecular biology). A transcription initiation region known to affect transcription efficiency may also be added to the promoter. A specific example of a promoter (T7 promoter) added to the 5' end of the primer when T7 RNA polymerase is used as the RNA polymerase for RNA amplification is an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 24.

To detect RNA contained in the novel coronavirus (SARS-CoV-2) using the oligonucleotide of the present invention, the following steps (1) to (6) may be carried out, for example.

(1) a step in which a second primer, which is an oligonucleotide capable of specifically hybridizing to the base sequence set forth in SEQ ID NOs: 13 to 23 or its complementary sequence, hybridizes to an RNA possessed by the novel coronavirus (SARS-CoV-2), and synthesizes a cDNA complementary to the specific base sequence by an enzyme having RNA-dependent DNA polymerase activity to generate an RNA-DNA double strand with the RNA;
(2) decomposing the RNA of the RNA-DNA double strand with an enzyme having ribonuclease H (RNase H) activity (production of single-stranded DNA);
(3) hybridizing a first primer, which is an oligonucleotide capable of specifically hybridizing to the base sequence of any one of SEQ ID NOs: 6 to 12 or its complementary sequence, to the single-stranded DNA (wherein either the first or second primer has a promoter for RNA polymerase added to its 5'-end side), and generating a double-stranded DNA containing a promoter capable of transcribing RNA of the base sequence of the amplified region or its complementary sequence by an enzyme having DNA-dependent DNA polymerase activity;
(4) producing an RNA transcription product using the double-stranded DNA as a template with an enzyme having RNA polymerase activity;
(5) a step of producing RNA transcription products in a chain reaction by using the RNA transcription products as templates for cDNA synthesis in the reaction of (1);
(6) A step of measuring the amount of the RNA transcription product over time using an oligonucleotide containing SEQ ID NO: 1 or its complementary sequence, the oligonucleotide being capable of hybridizing to the RNA of the novel coronavirus (SARS-CoV-2) or its complementary sequence and labeled with an intercalating fluorescent dye.

The enzyme having RNA-dependent DNA polymerase activity used in step (1), the enzyme having RNase H activity used in step (2), and the enzyme having DNA-dependent DNA polymerase activity used in step (3) can be added separately or in various combinations, but a reverse transcriptase derived from a retrovirus having both of the above activities can also be used. The reverse transcriptase is not particularly limited, but examples of the reverse transcriptase that can be used include AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molony Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, and HIV (Human Immunodeficiency Virus) reverse transcriptase, which are commonly used in the field of molecular biology.

The reaction temperature in the method for detecting RNA possessed by the novel coronavirus (SARS-CoV-2) according to the above-mentioned embodiment depends on the heat resistance and activity of each enzyme used, as well as the Tm of the primers/probes, etc., but when the enzymes used are AMV reverse transcriptase and T7 RNA polymerase, and the length of the primers/probes is in the range of 15 to 20 bases, the reaction temperature can be set in the range of 35 to 65°C, and more preferably in the range of 40 to 50°C.

The method for detecting RNA contained in the novel coronavirus (SARS-CoV-2) according to the above-mentioned aspect measures the fluorescence intensity over time, so that the measurement can be terminated at any time when a significant increase in fluorescence is observed, and the nucleic acid amplification and measurement can usually be completed within 20 minutes.

The method for detecting RNA possessed by the novel coronavirus (SARS-CoV-2) according to the above-mentioned aspect can automatically amplify and detect the RNA possessed by the novel coronavirus (SARS-CoV-2) by adding a sample to a reagent for detecting the novel coronavirus (SARS-CoV-2) RNA, which contains the above-mentioned first primer, second primer, and oligonucleotide labeled with an intercalating fluorescent dye, and placing the sample on a temperature-controlled block capable of detecting fluorescence over time. The oligonucleotide for detecting the RNA possessed by the novel coronavirus (SARS-CoV-2) of the present invention preferably consists of the base sequences shown in SEQ ID NOs: 2 to 4.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of oligonucleotides labeled with fluorescent intercalating dyes

The oligonucleotide labeled with an intercalating fluorescent dye (hereinafter referred to as the INAF probe) shown below in (A) was prepared based on the method disclosed in JP 2000-316587 A.

(A) A base sequence shown in SEQ ID NO: 2 (a base sequence from base 29212 to base 29228 of GenBank No. MN908947.1) or its complementary sequence, a base sequence shown in SEQ ID NO: 3 (a base sequence from base 29216 to base 29235 of GenBank No. MN908947.1) or its complementary sequence, a base sequence shown in SEQ ID NO: 4 (a base sequence from base 29228 to base 29245 of GenBank No. MN908947.1) or its complementary sequence, a base sequence shown in SEQ ID NO: 5 (a base sequence from base 29216 to base 29235 of GenBank No. MN908947.1) or its complementary sequence, No. MN908947.1) or its complementary sequence, and is labeled with thiazole orange via a linker at the 12th thymine from the 5' end in SEQ ID NO:2, the 8th thymine from the 5' end in SEQ ID NO:3, the 8th cytosine from the 5' end in SEQ ID NO:4, and the 5th cytosine from the 5' end in SEQ ID NO:5.

Example 2: Examination of oligonucleotides for detecting the new coronavirus (SARS-CoV-2)

The evaluation was performed by the following method using the combination of the first primer, second primer, and INAF probe (hereinafter referred to as the oligonucleotide combination) shown in Table 1. The INAF probe shown in Table 1 is the probe prepared in Example 1.

(1) A commercially available control reagent (AMPLIRUN SARS-CoV-2 RNA CONTROL, Vircell) was used as the RNA for the novel coronavirus (SARS-CoV-2). The control RNA was diluted to 1,000 copies/15 μL using RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.02% sodium cholate), and these were used as the RNA samples.

(2) A reaction solution having the following composition was dispensed into an evaporation drying tube and evaporated to dryness.
Composition of reaction solution: Concentration of RNA sample, starting solution, final concentration after addition (in 30 μL) 60 mM Tris-HCl buffer (pH 8.35)
300 mM trehalose 0.39 mM each dATP, dCTP, dGTP, dTTP
2.1mM each ATP, CTP, UTP
1.5mM GTP
3.2 mM ITP
0.4 μM first primer 0.4 μM second primer (an oligonucleotide having a T7 promoter (SEQ ID NO: 24) added to the 5' end of the oligonucleotide consisting of the base sequence of each SEQ ID NO)
10 nM INAF probe (prepared in Example 1)
0.025mg/mL bovine serum albumin 200U T7 RNA polymerase 9.0U AMV reverse transcriptase

(3) After the above evaporation and drying, 15 μL of the RNA sample was added and incubated at 46° C. for 5 minutes, after which 15 μL of a starting solution having the following composition was added and stirred.
Composition of enzyme solution: Final concentration during reaction (in 30 μL) 9.00% dimethyl sulfoxide 21 mM magnesium chloride 137 mM potassium chloride

(4) The evaporative drying tube was then reacted at 46°C using a thermostatic spectrophotometer capable of direct measurement, and the fluorescence intensity of the reaction solution was measured over time for 20 minutes.

The time when the starting solution was added and stirring was completed was set to 0 minutes. If the fluorescence intensity ratio of the reaction solution (the fluorescence intensity value at a specified time divided by the background fluorescence intensity ratio) exceeded 1.5, it was determined to be positive, and the time at that point was defined as the detection time. The results are shown in Tables 1, 2, and 3. The experiment was performed twice for each combination, and the average value was used. "N.D." means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.5 or less (negative determination).

Regarding the detection performance of SARS-CoV-2 RNA (Table 1), of the oligonucleotide combinations examined in this example (set 1 to set 21), all except sets 10-13, 15, and 18 detected 1,000 copies/test of SARS-CoV-2 RNA within 20 minutes. In particular, oligonucleotide combinations set 1-3, set 5-8, set 14, set 17, and 20 detected 1,000 copies/test of SARS-CoV-2 RNA within 10 minutes on average, and can be said to be oligonucleotide combinations capable of rapidly detecting SARS-CoV-2 RNA.

Among the oligonucleotide combinations examined in this example, set 2 and set 6 were selected from those with detection times of 10 minutes or less, and assay sensitivity evaluation (Table 2) and cross-reactivity evaluation with other coronavirus RNA (Table 3) were performed using these combinations.

The measurement sensitivity of the novel coronavirus (SARS-CoV-2) RNA was such that 30 copies/test of novel coronavirus (SARS-CoV-2) RNA was detected, and the cross-reactivity did not detect 2003 SARS (SARS-CoV), MERS, or coronaviruses (229E, OC43), showing high specificity for the novel coronavirus (SARS-CoV-2). The combination of oligonucleotides examined in this example can be said to be a combination of oligonucleotides capable of specifically detecting the RNA of the novel coronavirus (SARS-CoV-2).

Claims (8)

An oligonucleotide and a set of primers for detecting SARS-CoV-2, comprising :
The primer set includes a first primer that is an oligonucleotide having a base sequence set forth in any one of SEQ ID NOs: 7, 8, and 10, and a second primer that is an oligonucleotide having a base sequence set forth in any one of SEQ ID NOs: 14, 17, 19, 20, 21, and 23,
The detection oligonucleotide has a sequence selected from the base sequence set forth in any one of SEQ ID NOs: 2, 3, and 4, or a complementary sequence thereof.
A detection oligonucleotide and a set of primers . The detection oligonucleotide and a pair of primer set described in claim 1, characterized in that the detection oligonucleotide is an oligonucleotide labeled with a fluorescent dye and configured to change its fluorescent properties when it forms a complementary double-stranded strand. 3. The set of detection oligonucleotide and primer according to claim 2, wherein the detection oligonucleotide is labeled with an intercalative fluorescent dye. The detection oligonucleotide and a pair of primer sets according to claim 1, wherein the combination of the first primer and the second primer is a primer set that is an oligonucleotide consisting of a base sequence set forth in any one of SEQ ID NOs: 8 and 20, 10 and 20, 10 and 14, 10 and 17, 10 and 19, 10 and 21, or 10 and 23. A method for detecting the 2019 novel coronavirus (SARS-CoV-2), comprising using the detection oligonucleotide and a pair of primer sets described in claim 1. The detection method according to claim 5, characterized in that the detection oligonucleotide according to claim 2 is used. The detection method according to claim 6 , wherein the detection oligonucleotide is labeled with an intercalative fluorescent dye. The detection method according to claim 5, characterized in that the combination of the first primer and the second primer uses the primer set according to claim 4.