Methods and reagents for detecting novel coronavirus (SARS-CoV-2) RNA

Abstract

To provide detection methods in which the 2019 novel coronavirus (SARS-CoV-2) present in a sample is specifically amplified and false positives are less likely to occur.SOLUTION: Provided is a method for detecting a specific base sequence in a novel coronavirus (SARS-CoV-2) RNA, comprising: amplifying a nucleic acid consisting of a specific base sequence with a primer set consisting of at least a first primer and a second primer; and detecting the amplified nucleic acid with a nucleic acid probe labeled with a detectable label; the method for detecting a specific base sequence in a novel coronavirus (SARS-CoV-2) RNA characterized by that the first primer consists of a sequence of 20 or more consecutive bases selected from a specific base sequence or its complementary sequence, and that the second primer consists of a sequence of 20 or more consecutive bases selected from another specific base sequence or its complementary sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method and reagent for rapid, sensitive and specific detection of the 2019 novel coronavirus (hereinafter referred to as SARS-CoV-2) in a sample. [Background technology]

[0002] The novel coronavirus disease (hereafter referred to as COVID-19) is caused by the SARS-CoV-2 virus and was first identified 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).

[0003] Coronaviruses are enveloped, positive-sense, single-stranded RNA (ribonucleic acid) viruses with diameters of 80 to 160 nm that infect humans and a variety of animals, including dogs, pigs, cattle, and camels. Based on genetic characteristics, they are classified into genera: α, β, γ, and δ. Viruses that primarily 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 viruses account for 10 to 15% of cold cases (35% during epidemics). In addition to these, the SARS (severe acute respiratory syndrome) coronavirus and the MERS (Middle East respiratory syndrome) coronavirus were identified in 2003 and 2012, respectively. Both of these viruses cause severe infections, primarily pneumonia, in humans, with extremely high case fatality rates, approximately 10% for the former and over 30% for the latter (Non-Patent Document 1). SARS-CoV-2 belongs to the same βcoronavirus family as SARS and MERS. Based on full-nucleotide sequence analysis and phylogenetic tree analysis, SARS-CoV-2 shares 75-80% homology with SARS coronaviruses and 85-88% homology with bat coronaviruses. Therefore, SARS-CoV-2 is thought to have originated from a bat virus.

[0004] Initial symptoms are mild, including fever, fatigue, dry cough, loss of appetite, muscle pain, difficulty breathing, phlegm, and sore throat. Smell and taste disorders may also occur, and pneumonia may develop if the condition worsens. Initial symptoms usually last for 5 to 7 days and will gradually improve if they do not worsen.

[0005] If the condition worsens, breathing becomes difficult. In addition to pneumonia, inflammation of other respiratory organs, such as upper respiratory tract infection and bronchitis, may also occur. Even if severe pneumonia occurs, patients will gradually recover by receiving 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.

[0006] With typical viral infections, the likelihood of transmitting the virus to others is highest when symptoms are most pronounced. However, it is believed that COVID-19 patients can be infected when they are asymptomatic, at a mild stage, or at a severe stage. Identifying asymptomatic carriers of the virus, who are capable of infecting others despite showing no symptoms, is particularly important for preventing the spread of infection and hospital-acquired infections. Furthermore, large-scale screening tests are considered effective in identifying asymptomatic individuals, and require a test method with sufficient processing capacity and high sensitivity.

[0007] Highly sensitive testing methods include genetic testing, which amplifies and detects 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 operator to obtain reliable results. Furthermore, it takes time to obtain results and is not fast. On the other hand, antigen-antibody testing is simple, has high throughput, and is fast, but its sensitivity is lower than that of genetic testing, and there is a possibility of false negatives.

[0008] The TRC method used in the present invention (Patent Documents 2 and 3) is commercially available with an integrated device that handles everything from purification to detection, and is sufficiently rapid, providing results in about an hour. Furthermore, its sensitivity and specificity compare favorably with other nucleic acid amplification detection methods.

[0009] Because the nucleic acid to be amplified for the novel coronavirus (SARS-CoV-2) shares high base sequence homology with other coronaviruses, it has been extremely difficult to design primer sets and oligonucleotide probes that can detect its genes 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 amplification methods that can amplify RNA at relatively low, constant temperatures (e.g., 40°C to 50°C), the nucleic acid to be amplified is prone to forming higher-order structures, making designing the relevant primer sets and oligonucleotide probes even more difficult. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-131174 [Patent Document 2] Japanese Patent Application Laid-Open No. 2000-14400 [Patent Document 3] Japanese Patent Application Laid-Open No. 2001-37500 [Non-patent literature]

[0011] [Non-Patent Document 1] 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. Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to provide a method for highly sensitive, rapid, and specific amplification and detection of specific nucleic acid sequences in novel coronavirus (SARS-CoV-2) RNA present in a sample, and reagents for use therein. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to solve the above problems and have completed the present invention.

[0014] (1) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA, [i] amplifying a nucleic acid consisting of a specific base sequence using a primer set consisting of at least a first primer and a second primer; [ii] detecting the amplified nucleic acid with a nucleic acid probe labeled with a detectable label; A method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA, characterized in that the first primer consists of a sequence of 20 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 1 or its complementary sequence, and the second primer consists of a sequence of 20 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 2 or its complementary sequence. (2) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA described in (1), characterized in that the first primer consists of 20 consecutive bases of the base sequence described in SEQ ID NO: 1, and the second primer consists of 20 to 25 consecutive bases of SEQ ID NO: 2. (3) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA described in (2), characterized in that the first primer is selected from the base sequence set forth in any one of SEQ ID NOs: 3 to 15, and the second primer is selected from the base sequence set forth in any one of SEQ ID NOs: 17 to 22. (4) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA described in any one of (1) to (3), characterized in that the labeled nucleic acid probe is labeled with a fluorescent dye and is configured so that its fluorescent properties change when it forms a complementary double strand. (5) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA according to (4), characterized in that the fluorescent dye is an intercalating fluorescent dye. (6) A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA described in any one of (1) to (5), characterized in that the labeled nucleic acid probe consists of a sequence of 17 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 23 or its complementary sequence. (7) The step of amplifying the nucleic acid comprises: [i] the first primer and the second primer have a promoter sequence added to the 5' end of at least one of them, which allows transcription by RNA polymerase to be initiated; [ii] generating double-stranded DNA having a promoter region that can be transcribed by a primer set consisting of at least the first primer and the second primer, RNA-dependent DNA polymerase activity, RNase H activity, and DNA-dependent DNA polymerase activity; [iii] carrying out a transcription reaction using an RNA polymerase with the double-stranded DNA as a template to produce RNA consisting of a specific base sequence or its complementary sequence; [iv] The RNA serves as a template for generating the double-stranded DNA of [ii], and steps [ii] to [iii] are repeated. A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA according to any one of (1) to (6).

[0015] (8) A reagent for carrying out a detection process comprising amplifying a nucleic acid consisting of a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA using a primer set consisting of at least a first primer and a second primer, and detecting the amplified nucleic acid with a nucleic acid probe labeled with a detectable label, the reagent for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA comprising at least a first primer consisting of the sequence set forth in SEQ ID NO: 13 and a second primer consisting of the base sequence set forth in SEQ ID NO: 22. [Effects of the Invention]

[0016] The method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA of the present invention provides a primer set consisting of a first primer and a second primer for amplifying the specific base sequence, a nucleic acid amplification method, and a nucleic acid detection probe, thereby providing a method for rapid, highly sensitive, and specific detection of novel coronavirus (SARS-CoV-2) RNA.

[0017] The novel coronavirus (SARS-CoV-2) detection method of the present invention targets a single genetic region and can rapidly and sensitively detect the novel coronavirus (SARS-CoV-2) contained in a sample, while not detecting other coronavirus species, resulting in extremely low false positives and high specificity. This allows test results to be presented to physicians promptly, which is effective in preventing the spread of infection and determining appropriate treatment plans, contributing to the early improvement of symptoms and public health. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below.

[0019] In the present invention, the sample includes nasopharyngeal swabs, saliva, oral specimens, nasal specimens, sputum, bronchoalveolar lavage fluid, endotracheal aspirate, pleural effusion, blood, urine, feces, etc. Recently, in consideration of the safety of specimen collectors, application to saliva specimens has been desired.

[0020] The specific base sequence in the present invention is a sequence in the SARS-CoV-2 RNA sequence that consists of a sequence homologous to the first primer of the present invention, a sequence complementary to the second primer, and a sequence sandwiched between these, or a sequence consisting of a complementary sequence thereof.

[0021] The first primer and the second primer in the present invention consist of a sequence of 20 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 1 or its complementary sequence, and the second primer consists of a sequence of 20 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 2 or its complementary sequence, preferably consisting of 20 consecutive bases of the base sequence set forth in SEQ ID NO: 1 and the second primer consists of 20 to 25 consecutive bases of SEQ ID NO: 2, more preferably the first primer consists of a base sequence selected from the base sequence set forth in any of SEQ ID NO: 3 to SEQ ID NO: 15, and the second primer consists of a base sequence selected from the base sequence set forth in any of SEQ ID NO: 17 to SEQ ID NO: 22, and most preferably the first primer consists of the sequence set forth in SEQ ID NO: 13, and the second primer consists of the base sequence set forth in SEQ ID NO: 22.

[0022] It goes without saying that the above base sequences can be applied even if base mutations, deletions, additions, or chemical modifications are made to the sequences, as long as the specificity of hybridization with the target nucleic acid is not altered. Generally, if there is no difference in hybridization efficiency with the target nucleic acid under sterile conditions, for example, at 42°C in 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, it can be considered that there is no difference in hybridization specificity.

[0023] At least one of the first and second primers may have a promoter sequence added to the 5' end that contributes to the initiation of transcription by RNA polymerase. The promoter may be a promoter compatible with the RNA polymerase used in in vitro transcription reactions (e.g., T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, which are commonly used in the field of molecular biology). The promoter may also be further added with a transcription initiation region known to affect transcription efficiency. When T7 RNA polymerase is used as the RNA polymerase for RNA amplification, a specific example of a promoter (T7 promoter) added to the 5' end of the primer is an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 28.

[0024] The nucleic acid probe of the present invention is not particularly limited as long as it is labeled with a substance that exhibits a detectable signal. However, for versatility, it is preferably labeled with a fluorescent substance. Even more preferred are probes configured to change their fluorescent properties upon formation of a complementary double strand. Examples of probes configured to change their fluorescent properties include TaqMan probes, BEACON probes, and Q probes that utilize FRET (fluorescence resonance energy transfer). Although not particularly limited, intercalating fluorescent dye-labeled nucleic acid probes are the most preferred. One embodiment of the intercalating fluorescent dye-labeled nucleic acid probe is a nucleic acid probe labeled with an intercalating fluorescent dye via a suitable linker at least at the 3' end, 5' end, phosphodiester moiety, or base moiety of an oligonucleotide that can specifically hybridize to at least a portion of a specific base sequence of novel coronavirus (SARS-CoV-2) RNA or its complementary sequence. The intercalative fluorescent dye to be used for labeling is not particularly limited, and may be appropriately selected from commonly used fluorescent dyes such as oxazole yellow, thiazole orange, ethidium bromide, hemicyanine, etc., and their derivatives, taking into consideration their fluorescence intensity and fluorescence properties. Except when labeling the 3'-end with a fluorescent dye, the 3'-end of the oligonucleotide should preferably be appropriately modified with glycolic acid or the like to prevent nucleic acid elongation reaction from that end.

[0025] Furthermore, the nucleic acid probe of the present invention preferably consists of a sequence of 17 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 23 or its complementary sequence, and more preferably consists of the sequence set forth in SEQ ID NO: 25. As in the case of primers, base mutations, deletions, additions, or chemical modifications can also be made to the probe, as long as the specificity of the probe in hybridization with the target nucleic acid is not affected.

[0026] Methods for amplifying nucleic acids in the present invention include RT-PCR, NASBA (Nucleic Acid Sequence Based Amplification), and TMA (Transcription-Mediated Amplification), which use at least one primer set. Although not particularly limited, TRC (Transcription-Reverse Transcription Concerted Reaction), which can be performed at a constant temperature in approximately 20 minutes, is preferred.

[0027] A preferred embodiment of amplifying nucleic acids using the TRC method includes the following steps: (1) a second primer (having an RNA polymerase promoter sequence added to the 5' end) hybridizes to a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA, and an enzyme with RNA-dependent DNA polymerase activity synthesizes cDNA complementary to the specific base sequence, generating an RNA-DNA double-stranded strand with the RNA; (2) an enzyme with ribonuclease H (RNase H) activity degrades the RNA strand of the RNA-DNA double-stranded strand to generate single-stranded DNA; (3) a first primer hybridizes to the novel coronavirus (SARS-CoV-2) and an enzyme with DNA-dependent DNA polymerase activity generates double-stranded DNA containing a promoter capable of transcribing RNA of the specific base sequence or its complementary sequence; (4) an enzyme with RNA polymerase activity produces an RNA transcript using the double-stranded DNA as a template; and (5) the RNA transcript serves as a template for cDNA synthesis in the reaction (1), thereby generating RNA transcripts in a chain reaction.

[0028] Furthermore, when the nucleic acid amplification step is carried out in the presence of the intercalating fluorescent dye-labeled nucleic acid probe of the present invention, the nucleic acid amplification step and the detection step can be carried out simultaneously, and the fluorescence intensity corresponding to the amount of RNA transcription product can be monitored in real time.

[0029] The enzymes having RNA-dependent DNA polymerase activity, RNase H activity, and DNA-dependent DNA polymerase activity can be added individually or in various combinations, or a reverse transcriptase derived from a retrovirus that combines all three activities can be used. The reverse transcriptase is not particularly limited, and 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.

[0030] The reaction temperature in the method for detecting a specific base sequence of novel coronavirus (SARS-CoV-2) RNA according to the above embodiment depends on the heat resistance and activity of each enzyme used, as well as the Tm of the primers / probes, etc. However, if the enzymes used are AMV reverse transcriptase and T7 RNA polymerase and the length of the primers and probes is in the range of 15 to 25 bases, a constant reaction temperature can be set in the range of 35 to 65°C.

[0031] According to the above-described embodiment, it is possible to determine the specific base sequence of the novel coronavirus (SARS-CoV-2) RNA under optimized conditions, with nucleic acid amplification and measurement combined, within 20 minutes.

[0032] The method for detecting a specific base sequence of novel coronavirus (SARS-CoV-2) RNA according to the above embodiment involves adding a sample to a novel coronavirus (SARS-CoV-2) RNA detection reagent containing the aforementioned first primer, second primer, and an oligonucleotide labeled with an intercalating fluorescent dye, and placing the sample on a temperature-controlled block at a constant temperature that allows fluorescence detection over time.This method can automatically amplify and detect the RNA contained in the novel coronavirus (SARS-CoV-2), making it suitable for automation and useful for simplifying and speeding up operations.

[0033] The reagent for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA in the present invention comprises a primer set consisting of at least a first primer and a second primer, and in a preferred embodiment, comprises the primer set, an intercalative fluorescent dye-labeled nucleic acid probe, an enzyme with RNA-dependent DNA polymerase activity, an enzyme with RNase H activity, an enzyme with DNA-dependent DNA polymerase activity, an RNA polymerase, a substrate, a buffer, salts, etc. [Example]

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] Example 1: Preparation of oligonucleotides labeled with intercalating fluorescent dyes

[0036] An intercalative fluorescent dye-labeled oligonucleotide (hereinafter referred to as a probe) consisting of an oligonucleotide of SEQ ID NO: 24 or SEQ ID NO: 25, in which the 8th thymine from the 5' end in SEQ ID NO: 24 and the 12th thymine from the 5' end in SEQ ID NO: 25 were labeled with thiazole orange via a linker, was prepared according to the method disclosed in JP 2000-316587 A.

[0037] Example 2: Examination of oligonucleotides for detecting the novel coronavirus (SARS-CoV-2)

[0038] Evaluation was carried out by the method described below using the combinations of the first primer, second primer, and probe shown in Table 1. The INAF probes shown in Table 1 were the probes prepared in Example 1.

[0039] (1) For the novel coronavirus (SARS-CoV-2) RNA, a commercially available control reagent (Vircell, AMPLIRUN SARS-CoV-2 RNA CONTROL) was used (hereinafter referred to as standard RNA). The control RNA was diluted to 30 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 RNA samples.

[0040] (2) The reaction solution having the following composition was dispensed into tubes for evaporation and drying, and evaporated to dryness. Composition of reaction solution: Concentration of RNA sample, starting solution, final concentration after addition (in 30 μL) 60mM Tris-HCl buffer (pH8.35) 300mM trehalose 0.39mM each dATP, dCTP, dGTP, dTTP 2.1mM each of ATP, CTP, and UTP 1.5mM GTP 3.2mM ITP 0.4 μM first primer 0.4 μM second primer (an oligonucleotide consisting of the base sequence of each SEQ ID NO. with a T7 promoter (SEQ ID NO: 24) attached to the 5' end) 10 nM probe (prepared in Example 1) 0.025mg / mL bovine serum albumin 200U T7 RNA polymerase 9.0U AMV reverse transcriptase

[0041] (3) After the evaporation and drying, 15 μL of the RNA sample was added using an automatic genetic testing device TRCReady-80 (manufactured by Tosoh Corporation), and the mixture was kept at 46°C for 5 minutes. After that, 15 μL of a starting solution consisting of the following composition was added and stirred. Composition of enzyme solution: Final concentration during reaction (in 30 μL) 9.00% dimethyl sulfoxide 21mM magnesium chloride 137mM potassium chloride

[0042] (4) Subsequently, the reaction tube was reacted at 46° C. using an automated genetic testing device TRCReady-80 (manufactured by Tosoh Corporation), and the fluorescence intensity of the reaction solution was measured over time for 20 minutes.

[0043] The time when the starting solution was added and stirring was completed was designated as 0 minutes, and a positive result was determined when the fluorescence intensity ratio of the reaction solution (the fluorescence intensity value at a specified time divided by the background fluorescence intensity ratio) exceeded 1.6, and the time at this point was designated the detection time. The results are shown in Table 1. The test was performed twice for each combination. "ND" means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.6 or less (negative result).

[0044] Regarding the detection performance of novel coronavirus (SARS-CoV-2) RNA (Table 1), all of the oligonucleotide combinations examined in this example (Sets 1 to 14) detected 30 copies / test of novel coronavirus (SARS-CoV-2) RNA within 20 minutes. In particular, Sets 1 to 13 were detected within 10 minutes, demonstrating that these oligonucleotide combinations are capable of rapid detection of novel coronavirus (SARS-CoV-2) RNA. Note that Set 14 is a combination designed in the same gene region and is not applicable to the present invention.

[0045] [Table 1]

[0046] Example 3: Examination of oligonucleotides for detecting the novel coronavirus (SARS-CoV-2) using saliva spike samples

[0047] The effect of the matrix simulating a saliva sample was evaluated by the method described below using the combinations of the first primer, second primer, and probe shown in Tables 2 and 3. The probes listed in the tables were the probes prepared in Example 1.

[0048] (1) A saliva sample negative for the novel coronavirus (SARS-CoV-2) was centrifuged, and the supernatant was diluted 2-fold with PBS to obtain a 200 μL sample. Nucleic acid purification was performed using a TRCR Nucleic Acid Purification Kit (Tosoh Corporation). 200 μL of the sample was added to the denaturing reagent of the TRCR Nucleic Acid Purification Kit (Tosoh Corporation). After stirring, either (i) 3,000 copies or (ii) 1,000 copies of the standard RNA were added. Nucleic acid purification was performed using an automated genetic testing system, TRCReady-80 (Tosoh Corporation), to obtain 45 μL of purified nucleic acid, of which 15 μL was used as the RNA sample.

[0049] The operations (2) to (4) were carried out in the same manner as in Example 2.

[0050] The results for (i) 3000 copies added (equivalent to 1000 copies / test) are shown in Table 2, and the results for (ii) 1000 copies added (equivalent to 333 copies / test) are shown in Table 2. The time when the starting solution was added and stirring was completed was set to 0 minutes, and a positive result was determined when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a specified time by the background fluorescence intensity ratio) exceeded 1.6, and the time at this point was taken as the detection time. The results are shown in Table 1. The test was carried out twice for each combination. "ND" means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.6 or less (negative result).

[0051] The results of the SARS-CoV-2 (COVID-19) RNA detection performance using saliva spike samples (Tables 2 and 3) demonstrated that the oligonucleotide combinations in Sets 1 to 13 and Sets 16 to 21 were capable of detecting at least 1,000 copies / test of SARS-CoV-2 RNA within 20 minutes. These oligonucleotide combinations were suggested to be less susceptible to the effects of saliva sample matrix. In particular, Sets 11 and 21, consisting of the first primer (SEQ ID NO: 13) and second primer (SEQ ID NO: 22), detected 333 copies / test of SARS-CoV-2 RNA within 10 minutes, suggesting that these combinations are capable of rapid detection even when saliva is used as a sample. Note that Sets 14, 15, and 22 were designed in the same gene region and are not applicable to the present invention, but they may be affected by the matrix when applied to saliva samples. Possible influencing factors derived from saliva include various substances such as protein components and polysaccharides, but one possibility is nucleic acid components present in saliva, which are presumed to inhibit the reaction by interacting with the primers. Therefore, the combinations of Sets 1 to 13 and Sets 16 to 21 are thought to have little interaction with the influencing factors.

[0052] [Table 2]

[0053] [Table 3]

[0054] Among the oligonucleotide combinations examined in this example, set 11 was selected from those with a detection time of 10 minutes or less, and cross-reactivity assessment of other coronavirus RNAs was performed using this set (Table 3).

[0055] The procedure was the same as in Example 1 except for the RNA sample.

[0056] The cross-reactivity results are shown in Table 4. The test was performed three times. "ND" means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.6 or less (negative result). The 2003 SARS (SARS-CoV), MERS, and coronaviruses (229E, OC43) were not detected, and the primer combination tested in this example was highly specific for the novel coronavirus (SARS-CoV-2), demonstrating that it is possible to specifically detect the RNA of the novel coronavirus (SARS-CoV-2).

[0057] [Table 4]

Claims

1. A method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA, comprising: (i) amplifying a nucleic acid having a specific base sequence by the TRC method using a primer set consisting of at least a first primer and a second primer; (ii) detecting the amplified nucleic acid with a nucleic acid probe labeled with a detectable label; The first primer consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 15, and the second primer consists of the nucleotide sequence set forth in SEQ ID NO: 22; or A method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA, characterized in that the first primer consists of the base sequence set forth in SEQ ID NO: 13, and the second primer consists of the base sequence set forth in any of SEQ ID NOs: 17 to 22.

2. The first primer consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 15, and the second primer consists of the nucleotide sequence set forth in SEQ ID NO: 22; or The method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA according to claim 1, characterized in that the first primer consists of the base sequence set forth in SEQ ID NO: 13, and the second primer consists of the base sequence set forth in any one of SEQ ID NOs: 20 to 22.

3. The method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA according to any one of claims 1 to 2, characterized in that the labeled nucleic acid probe is labeled with a fluorescent dye and is configured so that its fluorescent properties change when it forms a complementary double strand.

4. The method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA according to claim 3, characterized in that the fluorescent dye is an intercalating fluorescent dye.

5. The method for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA according to any one of claims 1 to 4, characterized in that the labeled nucleic acid probe consists of a sequence of 17 or more consecutive bases selected from the base sequence set forth in SEQ ID NO: 23 or its complementary sequence.

6. the step of amplifying the nucleic acid comprises: (i) the first primer and the second primer have a promoter sequence added to the 5' end of at least one of them, which allows transcription initiation by RNA polymerase; (ii) generating double-stranded DNA having a promoter region that can be transcribed by a primer set consisting of at least the first primer and the second primer, an RNA-dependent DNA polymerase activity, an RNase H activity, and a DNA-dependent DNA polymerase activity; (iii) carrying out a transcription reaction using an RNA polymerase with the double-stranded DNA as a template to produce RNA consisting of a specific base sequence or its complementary sequence; (iv) the RNA serves as a template for generating the double-stranded DNA of (ii), and (ii) to (iii) are repeated. A method for detecting a specific base sequence in the novel coronavirus (SARS-CoV-2) RNA according to any one of claims 1 to 5.

7. A reagent for carrying out a detection process comprising at least a first primer and a second primer, the process comprising amplifying a nucleic acid comprising a specific base sequence in novel coronavirus (SARS-CoV-2) RNA by the TRC method using a primer set consisting of at least the first primer and the second primer, and detecting the amplified nucleic acid with a nucleic acid probe labeled with a detectable label, wherein the first primer consists of the base sequence set forth in any of SEQ ID NOs: 3 to 15, and the second primer consists of the base sequence set forth in SEQ ID NO: 22; or the first primer consists of the base sequence set forth in SEQ ID NO: 13, and the second primer consists of the base sequence set forth in any of SEQ ID NOs: 17 to 22.

8. The first primer consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 15, and the second primer consists of the nucleotide sequence set forth in SEQ ID NO: 22; or The reagent for detecting a specific base sequence in novel coronavirus (SARS-CoV-2) RNA according to claim 7, characterized in that the first primer consists of the base sequence set forth in SEQ ID NO: 13, and the second primer consists of the base sequence set forth in any one of SEQ ID NOs: 20 to 22.

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