Probe set for detecting SARS-cov-2 and uses thereof

Abstract

To provide a probe set for detecting SARS-CoV-2.SOLUTION: A probe set for detecting SARS-CoV-2 that may be contained in a specimen sample is disclosed, comprising the following fluorescently labeled probe (A) and the fluorescently labeled probe (B): (A) a fluorescently labeled probe containing a base sequence A1 shown in any of SEQ ID NOs: 3, 13, 14, 16 to 22, and 29, a base sequence A2 complementary to the base sequence A1, or a base sequence A3 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence A1 or A2, and labeled with a fluorescent dye; and (B) a fluorescently labeled probe containing a base sequence B1 shown in any of SEQ ID NOs: 2, 4 to 6, and 8 to 11, a base sequence B2 complementary to the base sequence B1, or a base sequence B3 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence B1 or B2, and labeled with a fluorescent dye.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a combination of oligonucleotides for detecting SARS-CoV-2 that may be contained in a specimen sample, etc. Furthermore, the present invention relates to a method for detecting SARS-CoV-2 that may be contained in a specimen sample using the combination of oligonucleotides, and a reagent / kit for use in the method. [Background technology]

[0002] SARS-CoV-2 is the virus that causes the novel coronavirus disease (COVID-19), which was first identified in Wuhan, Hubei Province, China in December 2019. COVID-19, caused by SARS-CoV-2, has caused a pandemic (global epidemic) since December 2019. As the global pandemic continues, mutant strains such as the Delta strain, which poses a high risk of severe symptoms, the highly infectious Omicron strain, and the Stealth Omicron strain have been reported.

[0003] Genetic, antigen, and antibody tests have been developed so far as testing methods for SARS-CoV-2. Of these, antigen tests have the advantage of being easy to use, but currently have problems with sensitivity. Furthermore, while antibody tests can confirm whether a person has had the disease in the past, they are not suitable for testing whether they are currently infected. In contrast, genetic tests have the advantage of being able to specifically detect SARS-CoV-2 with excellent sensitivity.

[0004] In genetic testing methods for RNA viruses such as SARS-CoV-2, the genomic RNA of SARS-CoV-2 is first reverse transcribed (RT) into cDNA using reverse transcriptase, and then the nucleic acid is amplified by PCR or other methods for detection. Currently, detection by RT-PCR mainly involves quantitative detection by performing real-time PCR using a double-labeled nucleic acid probe (also called a Taqman probe or hydrolysis probe, etc.) (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pathogen Detection Manual 2019-nCoV Ver.2.9.1 (March 19, 2020) National Institute of Infectious Diseases Summary of the Invention [Problem to be solved by the invention]

[0006] In the method of Non-Patent Document 1, it is possible to quantify the amount of virus based on a control RNA, but since photometry is required for each PCR cycle, detection takes about one hour even in the shortest case.

[0007] Melting curve analysis is known as a method for detecting nucleic acid amplification products. In melting curve analysis, nucleic acid amplification and detection can be performed in separate steps, and measurements can be performed relatively easily in as little as 30 minutes. Furthermore, melting curve analysis using a fluorescently labeled nucleic acid probe has the advantage of being easily adapted to genetic testing using an automatic analyzer. However, since melting curve analysis usually measures the fluorescence intensity in a predetermined detection temperature range, if a large number of mutations occur in the target nucleic acid region to which the probe binds, the melting temperature of the probe may drop significantly, resulting in incorrect detection.

[0008] RNA viruses such as SARS-CoV-2 are prone to frequent mutations. Even now, new mutant strains of SARS-CoV-2 continue to emerge. When performing amplification and detection of pathogenic microorganisms that are prone to frequent mutations using the RT-PCR method, many mutations occur in the sequence to which the probe or the like binds (anneals), as described above, making detection difficult. Therefore, in detecting RNA viruses that are prone to frequent mutations, a method can be considered in which multiple regions are amplified and detected simultaneously, so that even if one region cannot be amplified or detected, the other region can be amplified and detected, and even if a mutation occurs in either region, a positive judgment can be made. On the other hand, putting multiple primer and probe sets into the same reaction system increases the possibility of interactions such as forming dimers between oligonucleotides, and much trial and error is required to find the optimal primer and probe sequence. Therefore, it is not easy to design a probe set (a combination of multiple probes) that is less affected by some mutations in the target sequence and allows for highly sensitive detection.

[0009] In addition, in SARS-CoV-2 testing, RNA is typically extracted and purified from biological samples such as nasopharyngeal swabs, sputum, and saliva using an RNA extraction and purification kit, and the RNA extraction and purification liquid is then subjected to the next step, RT-PCR, etc. However, because RNA extraction and purification requires a great deal of effort and cost, there is a demand for a method that can detect SARS-CoV-2 with high sensitivity using biological samples prepared with only simple pretreatment.

[0010] The object of the present invention is to provide a useful probe set capable of detecting SARS-CoV-2, and further to provide a useful detection method for SARS-CoV-2 using the probe set. [Means for solving the problem]

[0011] As a result of intensive research to achieve the above object, the present inventors have found that SARS-CoV-2 can be detected easily, quickly, and with high sensitivity by using a probe set that combines labeled probes having specific base sequences. The present inventors have also found that the use of the probe set allows for high sensitivity detection even in the case of a mutant strain having many mutation sites, such as the Omicron strain of SARS-CoV-2. Based on this knowledge, the present inventors have further intensively researched and completed the present invention.

[0012] The present invention includes the following aspects. [Item 1] A probe set including the following fluorescently labeled probe (A) and the fluorescently labeled probe (B) used to detect SARS-CoV-2 that may be contained in a sample: (A) a fluorescently labeled probe comprising a base sequence A1 represented by any one of SEQ ID NOs: 3, 13, 14, 16 to 22, and 29, a base sequence A2 complementary to the base sequence A1, or a base sequence A3 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence A1 or A2, and which is labeled with a fluorescent dye; and (B) A fluorescently labeled probe comprising a base sequence B1 shown in any one of SEQ ID NOs: 2, 4 to 6, and 8 to 11, a base sequence complementary to base sequence B1, or a base sequence B3 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence B1 or B2, and which is labeled with a fluorescent dye. [Item 2] The probe set according to Item 1, wherein the fluorescently labeled probe (A) and the fluorescently labeled probe (B) are each labeled at only one of the 5' end and the 3' end. [Item 3] The probe set according to Item 1 or 2, wherein the fluorescent dye (A) is a fluorescent quenching dye that is quenched when the fluorescent labeled probe (A) is 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 fluorescent labeled probe, and the fluorescent dye (B) is a fluorescent quenching dye that is quenched when the fluorescent labeled probe (B) is 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 fluorescent labeled probe. [Item 4] The probe set according to any one of Items 1 to 3, wherein the fluorescent dye (A) and the fluorescent dye (B) are fluorescence quenching dyes that are quenched by interaction with guanine. [Item 5] The probe set according to any one of Items 1 to 4, wherein the fluorescent dye (A) and the fluorescent dye (B) are at least one fluorescent quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 6] The probe set according to any one of Items 1 to 5, wherein the fluorescent dye (A) and the fluorescent dye (B) are at least one fluorescent 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, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 7] The probe set according to any one of Items 1 to 6, wherein the labeled terminal base of both the fluorescently labeled probe (A) and the fluorescently labeled probe (B) is cytosine. [Item 8] A probe set according to any one of items 1 to 7, which is used to detect SARS-CoV-2 that may be contained in a biological specimen sample that has not been subjected to a nucleic acid purification process. [Item 9] A method for detecting SARS-CoV-2 that may be contained in a sample using a probe set described in any one of Items 1 to 8. [Item 10] The method described in Item 9, which comprises a step of detecting by RT-PCR-melting curve analysis. [Item 11] The method according to Item 10, wherein the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to Family B. [Item 12] The method according to Item 11, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Item 13] The method according to any one of Items 10 to 12, wherein the enzyme having reverse transcription activity used in the RT reaction is a reverse transcriptase derived from Moloney Murine Leukemia Virus (M-MLV) or a mutant thereof. [Item 14] The method according to any one of Items 9 to 13, wherein the specimen sample is a specimen sample derived from a living body that has not been subjected to a nucleic acid purification step. [Item 15] A reagent kit for detecting SARS-CoV-2 that may be contained in a sample, comprising the probe set described in any one of items 1 to 8. Effect of the Invention

[0013] The present invention provides a useful probe set capable of detecting SARS-CoV-2 that may be contained in a specimen sample. The probe set can detect SARS-CoV-2 easily, quickly, and with high sensitivity. The probe set can detect SARS-CoV-2 with high sensitivity, even when using a sample in which nucleic acid has not been highly isolated or purified, i.e., a sample from which RNA has been simply extracted. Furthermore, the probe set can detect SARS-CoV-2 with high sensitivity, even if SARS-CoV-2 has many mutations, such as the Omicron strain. The detection method, reagent, or kit using the probe set enables easy, rapid, accurate, and highly sensitive detection of SARS-CoV-2, which can greatly contribute to the field of clinical diagnosis. [Brief description of the drawings]

[0014] [Figure 1] Fig. 1 shows a representative example of the results of Test Example 1. (A graph showing the detection results when melting curve analysis was performed using a combination of nucleic acid probes of SEQ ID NO: 1 and SEQ ID NO: 2.) [Diagram 2] Fig. 1 shows a representative example of the results of Test Example 1. (A graph showing the detection results when melting curve analysis was performed using a combination of the nucleic acid probes of SEQ ID NO: 3 and SEQ ID NO: 2.) [Diagram 3]Fig. 1 shows a representative example of the results of Test Example 1. (A graph showing the detection results when melting curve analysis was performed using a combination of nucleic acid probes of SEQ ID NO: 3 and SEQ ID NO: 4.) [Figure 4] Fig. 1 shows a representative example of the results of Test Example 1. (Graph showing the detection results when melting curve analysis was performed using a combination of the nucleic acid probes of SEQ ID NO: 29 and SEQ ID NO: 2.) [Diagram 5] Fig. 1 shows a representative example of the results of Test Example 2. (A graph showing the detection results when melting curve analysis was performed using a combination of the nucleic acid probes of SEQ ID NO: 4 and SEQ ID NO: 3.) [Figure 6] Fig. 1 shows the results of Test Example 2. (Graph showing the detection results when melting curve analysis was performed using a combination of nucleic acid probes of SEQ ID NO: 6 and SEQ ID NO: 3.) [Figure 7] Fig. 1 shows a representative example of the results of Test Example 3. (Graph showing the detection results when melting curve analysis was performed using a combination of nucleic acid probes of SEQ ID NO: 3 and SEQ ID NO: 2.) [Figure 8] Fig. 1 shows the results of Test Example 3. (Graph showing the detection results when melting curve analysis was performed using a combination of nucleic acid probes of SEQ ID NO: 12 and SEQ ID NO: 2.) [Figure 9] Fig. 1 shows the results of Test Example 4. (Graph showing the detection results when melting curve analysis was performed using a combination of the nucleic acid probes of SEQ ID NO: 29 and SEQ ID NO: 2.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in further detail below with reference to embodiments of the present invention, but the present invention is not limited thereto. All non-patent and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" includes the concepts of "consisting essentially of" and "consisting only of".

[0016] In this specification, a nucleic acid primer may be simply referred to as a primer, and a nucleic acid probe, a labeled probe, and a fluorescently labeled probe may be simply referred to as a probe, and these are collectively referred to as oligonucleotides.

[0017] In one embodiment, the present invention provides a method for detecting SARS-CoV-2 by using a combination of labeled probes having specific base sequences. This method allows simple, rapid, highly sensitive, and accurate detection of SARS-CoV-2, even when detecting SARS-CoV-2 mutants with many mutations, such as the Omicron strain. In addition, according to the present invention, various SARS-CoV-2, including mutants such as the Omicron strain, can be reliably detected by melting curve analysis or the like, and can be detected with high sensitivity. The present invention includes an embodiment characterized in that a probe set is used that combines fluorescently labeled probes (also referred to as "nucleic acid probes" or the like) having specific base sequences that target two specific regions in the genomic RNA sequence of SARS-CoV-2.

[0018] [Methods for detecting SARS-CoV-2] In one embodiment, the method for detecting SARS-CoV-2 that may be contained in a specimen sample is a method using a combination of nucleic acid probes described below. The method may be a method for determining whether SARS-CoV-2 is present or absent in a specimen sample. The method may also be a method for quantifying SARS-CoV-2 contained in a specimen sample. In a specific embodiment, by using the probe set of the present invention, SARS-CoV-2 that may be contained in a specimen sample can be detected with high sensitivity without strain differences, for example, in RT-PCR-melting curve analysis.

[0019] In a specific embodiment, a method for detecting SARS-CoV-2 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 sample into cDNA; (2) generating one or more nucleic acid amplification products using the cDNA from step (1) as a template; and (3) detecting one or more nucleic acid amplification products of step (2) using a plurality of nucleic acid probes as described below; It is preferable that the method includes carrying out step (2) by a PCR reaction and carrying out step (3) by a melting curve analysis method (RT-PCR-melting curve analysis method). Steps (1), (2), and (3) may be carried out in the same reaction solution. In addition, two or more steps among steps (1), (2), and (3), for example, step (1) and step (2), may be carried out consecutively or simultaneously in parallel.

[0020] [Process (1)] In one embodiment, step (1) is preferably a step of performing a reverse transcription reaction using a nucleic acid primer and a reverse transcriptase to generate cDNA from a target RNA. The reverse transcriptase is not particularly limited as long as it is an enzyme having reverse transcription activity, and examples thereof include reverse transcriptase (RNA-dependent DNA polymerase) derived from M-MLV (Moloney Murine Leukemia Virus) and AMV (Avian Myeloblastosis Virus), and mutants thereof. Examples of the mutant include a mutant in which 1 to 3 amino acids are deleted, substituted, inserted, and / or added in the wild-type amino acid sequence, or a mutant showing 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 thereof include a mutant in which RNase H activity is deleted in order to increase the efficiency of cDNA synthesis. In addition, 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.

[0021] 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). The conditions for the reverse transcription reaction are not particularly limited as long as the reaction proceeds. The temperature and time of the reverse transcription reaction (or the set temperature and set time in the reverse transcription reaction device) 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.

[0022] [Process (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). The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level at which it can be visualized, i.e., to 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 inspection, and environmental inspection. 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 thereto.

[0023] (PCR reaction) PCR reaction is a reaction catalyzed mainly by DNA polymerase. PCR reaction usually includes three steps, namely (i) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (ii) annealing of a primer to a template single-stranded DNA, and (iii) extension of the primer using DNA polymerase, as one cycle, and the cycle is repeated. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to family B from the viewpoints of simplicity, speed, high sensitivity, and resistance to amplification inhibition by a sample. In addition, when step (3) is performed by melting curve analysis, it is preferable to use a DNA polymerase belonging to family B that does not have 5'→3' exonuclease activity from the viewpoint of using a fluorescence quenching probe.

[0024] The conditions of the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for about 0 to 300 seconds (for example, about 0.5 to 300 seconds), the second or subsequent (repeated) step (i) may be performed at 80 to 100°C for about 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for about 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for about 1 to 300 seconds. It is preferable that the cycle from step (i) to (iii) is repeated 30 to 70 times. The temperature and time of the repeated cycle may be changed every 1 to 3 cycles.

[0025] (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 DNA polymerase belonging to family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, and more preferably, includes DNA polymerases derived from bacteria of the genus Pyrococcus and Thermococcus. In addition, suitable DNA polymerases also include mutants thereof that do not lose DNA polymerase activity derived from Archea belonging to family B. Examples of the mutants include mutants in which 1 to 3 amino acids are deleted, substituted, inserted, and / or added in the wild-type amino acid sequence, or mutants that show 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 of DNA polymerase mutants include, but are not limited to, mutants aimed at enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, and the like.

[0026] Examples of 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.

[0027] Examples of 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. 9doN-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be particularly preferably used in the present invention from the viewpoint of excellent extensibility and thermostability.

[0028] PCR enzymes using these DNA polymerases are commercially available, and examples of such enzymes include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0029] (DNA polymerase derived from KOD) In the present specification, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to Thermococcus kodakaraensis-derived DNA polymerase and its variants (for example, KOD-derived DNA polymerase in which 3'→5' exonuclease activity has been deleted by substituting, deleting, inserting and / or adding 1 to 3 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, which is a DNA polymerase belonging to family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by inhibitors derived from samples. In the present invention, the use of such a KOD DNA polymerase is more preferable in terms of simple, rapid and highly sensitive detection of SARS-CoV-2, as shown in the examples described later.

[0030] (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 SARS-CoV-2 with which the probe set described below can form a complex. From the viewpoint of easily obtaining a more sensitive determination result, the nucleic acid primer set is preferably a nucleic acid primer set that can amplify a nucleotide sequence including a part or all of the nucleotide sequence of the SARS-CoV-2 genomic RNA shown in SEQ ID NOs: 27 and 28, which are also called the N1 region and the N2 region, respectively, in the SARS-CoV-2 genomic RNA.

[0031] For example, the nucleic acid primer set is a nucleic acid primer set capable of amplifying a part or all of the nucleotide sequence of the genome RNA of SARS-CoV-2 shown in SEQ ID NO: 27 and 28 to which the fluorescently labeled probes (A) and (B) described below can bind, respectively. The primer set is designed so that one primer is complementary to the DNA extension product of the other primer. For example, the nucleic acid primer set may be a nucleic acid primer set including a nucleic acid primer having a nucleotide sequence shown in SEQ ID NO: 23 and a nucleic acid primer having a nucleotide sequence shown in SEQ ID NO: 24, or a nucleic acid primer having a nucleotide sequence shown in SEQ ID NO: 25 and a nucleic acid primer having a nucleotide sequence shown in SEQ ID NO: 26, but is not limited thereto. For example, the nucleic acid primer set may be a combination of nucleic acid primers having nucleotide sequences complementary to the nucleotide sequences of SEQ ID NO: 23 and 24, respectively, or a combination of nucleic acid primers having nucleotide sequences complementary to the nucleotide sequences of SEQ ID NO: 25 and 26, respectively.

[0032] The primer set may also include a primer having a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence of each of the primers.

[0033] [Process (3)] The embodiment of step (3) is not particularly limited, and can be performed by any method known in the art. SARS-CoV-2 to be detected is prone to mutation, and various mutant genome sequences exist. Here, if a mismatch occurs between the base sequence of the primer or probe and the base sequence of the target SARS-CoV-2 mutant strain (Delta strain, Omicron strain, etc.), the binding strength of the primer or probe to the viral RNA or the nucleic acid amplification product derived therefrom (target nucleic acid) may decrease. In particular, if there are many mismatches between the target nucleic acid and the probe in the real-time RT-PCR method, the probe cannot sufficiently bind to the target nucleic acid, so the rise of the amplification curve may be delayed or may not rise at all, which may induce a false negative. In the case of the melting curve analysis method, since step (3) is performed after RT-PCR is completed, even if there is a mismatch in the primer or probe, detection is possible as long as the nucleic acid amplification product is finally obtained, so the effect of mismatches can be suppressed more than in the real-time RT-PCR method. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product by melting curve analysis. Furthermore, by detecting the 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).

[0034] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (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 a highly sensitive determination result in the detection of SARS-CoV-2, it is preferable to use a nucleic acid probe described below that can specifically react with the nucleic acid amplification product derived from SARS-CoV-2 in 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, such as, but not limited to, a temperature at least 5°C lower than the Tm value of the nucleic acid probe, more preferably at least 10°C lower.

[0035] (Nucleic acid probe) In the present invention, it is preferable to use, as the nucleic acid probe, a probe set including at least the following fluorescently labeled probe (A) and the following fluorescently labeled probe (B): (A) a fluorescently labeled probe comprising a base sequence A1 represented by any one of SEQ ID NOs: 3, 13, 14, 16 to 22, and 29, a base sequence A2 complementary to the base sequence A1, or a base sequence A3 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence A1 or A2, and which is labeled with a fluorescent dye; and (B) A fluorescently labeled probe comprising a base sequence B1 shown in any one of SEQ ID NOs: 2, 4 to 6, and 8 to 11, a base sequence B2 complementary to base sequence B1, or a base sequence B3 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence B1 or B2, and which is labeled with a fluorescent dye. Here, the fluorescently labeled probe (A) is a probe designed to bind to a base sequence (SEQ ID NO: 27) called the N1 region in the genomic RNA of SARS-CoV-2. The fluorescently labeled probe (B) is a probe designed to bind to a base sequence (SEQ ID NO: 28) called the N2 region in the genomic RNA of SARS-CoV-2. The fluorescently labeled probes (A) and (B) may be labeled with different fluorescent dyes, but are preferably labeled with the same fluorescent dye. By using such a set of probes, for example, SARS-CoV-2 can be detected with high sensitivity even in melting curve analysis, and SARS-CoV-2 mutants with many mutations, such as the Omicron strain, can also be detected with high sensitivity.

[0036] In a specific embodiment, the fluorescently labeled probe (A) and the fluorescently labeled probe (B) may be probes containing base sequences A3 and B3, respectively. In the base sequences A3 and B3, the number of bases that can be mutated (substituted, deleted, inserted or added) may preferably be 1 or 2. When a base mutation is present in this way, the probe is said to contain a mismatched base, and such a probe containing a mismatched base can also be suitably used in the present invention.

[0037] In this specification, containing a mismatched base (or simply referred to as "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after the nucleic acid amplification reaction, the base sequence of any single-stranded nucleic acid resulting from dissociation of the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., an adenine base, a cytosine base, a thymine base, and a universal base). For example, when detecting a region of the target nucleic acid that is prone to mutation, a mismatched base (e.g., a universal base) can be selected at the position of the probe that corresponds to the base that is prone to mutation.

[0038] 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 effect of the present invention. From the viewpoint of more reliable detection of the nucleic acid amplification product, it is preferable that the mismatched base is not the 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 an odd number, n / 2 when n is an even number), 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.

[0039] From the viewpoint of enabling more sensitive detection, the fluorescently labeled probe (A) is preferably a fluorescently labeled probe containing a base sequence A1 shown in any one of SEQ ID NOs: 3, 13, 14, 16 to 22, and 29 or a base sequence A2 complementary to the base sequence A1, and labeled with a fluorescent dye. Furthermore, from the viewpoint of enabling higher detection through a synergistic effect in combination with the fluorescently labeled probe of (B), the fluorescently labeled probe of (A) is preferably a fluorescently labeled probe containing the base sequence A11 shown in any one of SEQ ID NOs: 3, 13, 14, 16 to 18, and 29 or a base sequence A21 complementary to the base sequence A11 and labeled with a fluorescent dye, more preferably a fluorescently labeled probe containing the base sequence A12 shown in any one of SEQ ID NOs: 3, 16 to 18, and 29 or a base sequence A22 complementary to the base sequence A12 and labeled with a fluorescent dye, and particularly preferably a fluorescently labeled probe containing the base sequence A13 shown in SEQ ID NO: 3 or 29 or a base sequence A23 complementary to the base sequence A13 and labeled with a fluorescent dye.

[0040] From the viewpoint of enabling more sensitive detection, the fluorescently labeled probe (B) is preferably a fluorescently labeled probe containing a base sequence B1 shown in any one of SEQ ID NOs: 2, 4 to 6, and 8 to 11 or a base sequence B2 complementary to the base sequence B1, and labeled with a fluorescent dye. In a further specific embodiment, from the viewpoint of enabling higher detection in a synergistic manner together with the fluorescently labeled probe (A), the fluorescently labeled probe (B) is preferably a fluorescently labeled probe containing a base sequence B11 shown in SEQ ID NO: 6 or 8 or a base sequence B21 complementary to the base sequence B11, and labeled with a fluorescent dye. From another viewpoint, in order to prevent the detection peak waveform from being distorted and to facilitate more sensitive detection when detecting SARS-CoV-2 mutant strains with many mutations, such as the Omicron strain, the fluorescently labeled probe (B) is preferably a fluorescently labeled probe containing the base sequence B12 shown in SEQ ID NO: 2 or 4 or the base sequence B22 complementary to the base sequence B12 and labeled with a fluorescent dye, and in particular, is preferably a fluorescently labeled probe containing the base sequence B13 shown in SEQ ID NO: 2 or the base sequence B23 complementary to the base sequence B13 and labeled with a fluorescent dye.

[0041] The probe set of the present invention may be any combination of the fluorescently labeled probe (A) and the fluorescently labeled probe (B). Alternatively, one or more of the fluorescently labeled probes (A) may be combined with one or more of the fluorescently labeled probes (B). In a particular preferred embodiment, the probe set may be a combination of (A) a fluorescently labeled probe containing the base sequence A13 shown in SEQ ID NO: 3 or SEQ ID NO: 29 or a base sequence A23 complementary to the base sequence A13 and labeled with a fluorescent dye, and (B) a fluorescently labeled probe containing the base sequence B12 shown in SEQ ID NO: 2 or 4 or a base sequence B22 complementary to the base sequence B12 and labeled with a fluorescent dye.

[0042] The above-mentioned probes (A) and (B) included in the probe set of the present invention are preferably labeled with the same fluorescent dye, and may be labeled with the fluorescent dye in any manner. For example, each of the probes (A) and (B) may be labeled with a fluorescent dye at either or both of the 5'-end or 3'-end, but from the viewpoint of more reliably obtaining the effects of the present invention, it is preferable that each of the probes (A) and (B) is labeled with only either the 5'-end or the 3'-end. Here, the probes (A) and (B) do not necessarily have to be labeled at the same end, and one probe may be labeled at the 5'-end and the other probe may be labeled at the 3'-end.

[0043] In one embodiment, each of the probes (A) and (B) contained in the probe set of the present invention is preferably labeled with a fluorescent dye so as to cause quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows an identity of 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 to a base sequence complementary to the probe itself, and more preferably labeled with a fluorescent dye so as to cause quenching.

[0044] 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, and is particularly preferably a fluorescence quenching dye that quenches fluorescence by interaction with guanine when hybridized with a target nucleic acid amplification product. Specific examples of the fluorescent quenching dye include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of 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, 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, BODIPY-665).

[0045] More specifically, examples of the fluorescence quenching dye that is quenched by 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.

[0046] In a particular preferred embodiment, a probe in which the terminal base labeled with a fluorescence quenching dye is cytosine is more preferred. When such a probe hybridizes to a nucleic acid amplification product, it can form a base pair with a guanine base in the nucleic acid amplification product and interact with it to quench the fluorescence, making it very easy to measure the change in the fluorescence intensity of the reaction solution.

[0047] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe is hybridized, the fluorescence can be quenched if the guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the cytosine base of the probe (a base that forms a base pair with the cytosine base is considered to be 1).

[0048] Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in the fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. For example, when the probe is hybridized, quenching can be achieved if a guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[0049] In a particular preferred embodiment, the probe set of the present invention is used in step (3). By using the probe set of the present invention in this manner, even SARS-CoV-2 mutants containing many mutations, such as the Omicron strain, can be correctly detected.

[0050] 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 probe set 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 probe set 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) of step (2) at an endpoint. (3-c) after completion of the step (2), hybridizing the probe set 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. By the step (3-a), (3-b), or (3-c), the formation of a complex formed between the nucleic acid amplification product and the probe set can be detected simply, quickly, and with high sensitivity. The 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) can be performed. In one embodiment, from the viewpoint of more rapid detection of the nucleic acid amplification product, the step (3-b) or (3-c) is preferred. In particular, the step (3-c), i.e., the melting curve analysis method, is preferred.

[0051] (Step (3-a)) Step (3-a) is a method for monitoring the progress of a 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.

[0052] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the end point, thereby enabling rapid detection of the target nucleic acid that may be contained in the specimen. Furthermore, the approximate amount of the target nucleic acid can be estimated by comparing the fluorescence intensity at the end point. For example, the progress of the nucleic acid amplification reaction is monitored at an end point by measuring the fluorescence intensity of a reaction solution containing a set of nucleic acid probes labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured, and the fluorescence intensity of the reaction solution after the reaction is compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming the presence or absence of amplification of the target nucleic acid. Alternatively, the presence or absence of the target nucleic acid that may be contained in the specimen sample can also be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. The control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the specimen 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 convenient detection, it is preferable to measure it at an end point.

[0053] (Step (3-c)) In step (3-c), measuring the temperature dependency of the fluorescence intensity may 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 obtained by first differentiating the obtained fluorescence intensity with respect to temperature. The fluorescence intensity may also be converted into a fluorescence quenching rate or the like according to the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. In general, 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 is 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 of target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0054] As an example, melting curve analysis is also applied to SNP analysis. When there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases mismatch when the probe hybridizes, and the Tm value is generally low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0055] [Specimen] The specimen sample that can be used in the present invention is not particularly limited as long as it may contain SARS-CoV-2. Examples include, but are not limited to, oral scrapings, pharyngeal swabs, nasopharyngeal swabs, nasal swabs, nasal aspirates, sputum, bronchial lavage, alveolar lavage, saliva, etc. collected from subjects suspected of infection with SARS-CoV-2. When a biological sample is used as the specimen sample to be measured, pretreatment such as dilution or suspension, centrifugation, enzyme treatment, or nucleic acid extraction may be performed depending on each biological sample, although this is not particularly limited.

[0056] The method of collecting and preparing the specimen is not particularly limited, and known methods can be used depending on the type, purpose, etc. of the specimen. In a particular preferred embodiment, the specimen does not need to be a sample from which RNA has been isolated or purified, and for example, a specimen collected from a living body may be subjected to proteolytic denaturation treatment with a protease (e.g., proteinase K) and / or heat treatment (e.g., at 60 to 100°C for 1 second to 10 minutes) to previously degrade and remove RNase (ribonuclease) and DNase (DNA degrading enzyme) present in the specimen, and the resulting sample may be used as is.

[0057] The method of nucleic acid extraction is not particularly limited, and a known method can be used depending on the type and purpose of the specimen. The nucleic acid extraction may be a method of extracting mainly RNA, or a method of extracting nucleic acid without distinguishing between RNA and DNA. For nucleic acid extraction, for example, a kit sold by each manufacturer may be used. For example, QIAamp Viral RNA Mini Kit (QIAGEN). An automatic extraction and purification device may also be used.

[0058] In a particular preferred embodiment, the specimen may be one in which the nucleic acid purification step, which is usually considered essential in conventional nucleic acid amplification reactions, has been omitted. In addition to the need for a dedicated reagent, nucleic acid purification has the problem that the work is complicated, time-consuming, and laborious. When a specimen without a nucleic acid purification step is used, the time from collection of the specimen to obtaining the genetic test result can be shortened, and for example, the time from collection of the specimen to obtaining the genetic test result can be within one day, preferably within half a day, more preferably within six hours, even more preferably within three hours, and particularly preferably within two hours (for example, within one hour). In this way, when the method of the present invention is performed using a specimen that has not undergone a nucleic acid purification step, the effort of nucleic acid purification can be omitted, and SARS-CoV-2 can be detected with high sensitivity while being simple and in a short time.

[0059] [Reagents for detecting SARS-CoV-2] In another embodiment, the present invention provides a reagent for detecting SARS-CoV-2. The reagent preferably contains at least components necessary for reverse transcription reaction, nucleic acid amplification reaction, and detection in addition to the probe set of the present invention described above. The necessary components can be those known in the art. 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 inorganic salts such as magnesium salts. The nucleic acid primer set for PCR, which also serves as a nucleic acid primer for reverse transcription, and the nucleic acid probe set for detection can contain multiple sets in order to amplify multiple regions of SARS-CoV-2. The concentration of each component can be appropriately adjusted, but for example, each of the nucleic acid probes contained in the nucleic acid probe set of the present invention is preferably used at 0.01 to 1 μM, and more preferably used at 0.02 to 0.5 μM. The amount of each nucleic acid probe contained in the probe set does not need to be the same, and the concentration of the fluorescently labeled probe (A) and the concentration of the fluorescently labeled probe (B) may be different. Each nucleic acid primer contained in the nucleic acid primer set is preferably at 0.01 to 10 μM. The reverse transcriptase is preferably at 0.01 to 10 U / μL, more preferably at 0.02 to 2 U / μL. The DNA polymerase is preferably at 0.01 to 1 U / μL, more preferably at 0.02 to 0.5 U / μL. The deoxyribonucleoside triphosphates (dNTPs) are preferably at 0.02 to 1 mM, more preferably at 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably at 0.1 to 10 mM, more preferably at 1 to 5 mM.

[0060] Furthermore, the reagent of the present invention may contain additives known in the art for the purpose of suppressing non-specific amplification and promoting the reaction. Examples of additives for suppressing non-specific amplification include known anti-DNA polymerase antibodies and phosphoric acid. 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, Nonidet (registered trademark) P40, and the like. In addition, in order to reduce degradation of the target genomic RNA of SARS-CoV-2, the reagent of the present invention may contain an inhibitor or suppressor of RNase (e.g., RNase inhibitor). In addition, in order to facilitate the determination of false negatives, it is also preferable that the reagent of the present invention 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.

[0061] [Reagent kit for detecting SARS-CoV-2] In another embodiment, the present invention provides a reagent kit for detecting SARS-CoV-2. The kit of the present invention is not particularly limited as long as it includes the probe set of the present invention or the reagent of the present invention described above and is configured to detect (including differentiation) SARS-CoV-2. For example, the kit of the present invention can optionally include a reagent capable of detecting (including quantification) the presence of the target substance, and / or an instruction manual for use that explains the method of use, etc. For example, the kit of the present invention can be provided in such a form that the probe set, the components necessary for the reverse transcription reaction, the components necessary for the nucleic acid amplification reaction, and the components necessary for the detection of the amplification product are sealed in the same container or in separate containers, for example, packaged in a single package, and includes information on the method of use of the kit. The kit of the present invention can also include a positive control solution and / or a negative control solution. EXAMPLES

[0062] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0063] [Test Example 1: Measurement of SARS-CoV-2 mutant strains using a combination of nucleic acid probes] (1) Sample preparation The samples used were nasopharyngeal swabs (positive samples) from subjects who were diagnosed with SARS-CoV-2 Delta or Omicron strain infection by genome analysis. First, 10 μL of proteinase K solution (Kanto Chemical) was dispensed into a 1.5 mL tube, and the nasopharyngeal swabs from each subject were added and mixed. Next, these mixtures were heated at 65°C for 5 minutes, then heated at 95°C for another 5 minutes, and the reaction solution was cooled sufficiently. The reaction solution was mixed with an equal amount of GeneCube dedicated dissolving solution (Toyobo) to prepare the specimen. Note that this sample was not subjected to nucleic acid purification treatment, so it corresponds to a sample containing contaminants derived from the sample. (2) Reverse transcription, nucleic acid amplification, melting curve analysis, and evaluation The above samples were added to the following reagents, and SARS-CoV-2 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis.

[0064] (reagent) The combination of SEQ ID NO: 1 and SEQ ID NO: 2, the combination of SEQ ID NO: 3 and SEQ ID NO: 2, the combination of SEQ ID NO: 3 and SEQ ID NO: 4, and the combination of SEQ ID NO: 29 and SEQ ID NO: 2 were prepared as probe sets, and the following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). The probe of SEQ ID NO: 29 has a nucleic acid sequence in which the adenine (A) corresponding to the 16th base of the probe of SEQ ID NO: 3 is changed to the universal base inosinic acid (I), and has a nucleic acid sequence in which SEQ ID NO: 3 is mutated by one base. The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were adjusted and used as described in the 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 was performed normally. 0.5 μM primer shown in SEQ ID NO: 23 3.0 μM primer shown in SEQ ID NO:24 0.25 μM probe shown in SEQ ID NO: 1, 3, or 29 (5' or 3' end labeled with BODIPY-FL) 0.5 μM primer shown in SEQ ID NO: 25 3.0 μM primer shown in SEQ ID NO:26 0.25 μM probe shown in SEQ ID NO: 2 or 4 (5' or 3' end labeled with BODIPY-FL)

[0065] (Reverse transcription, nucleic acid amplification and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (One cycle) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0066] (3) Results 1 to 4 show detection graphs obtained when detecting RNA extracted samples of each SARS-CoV-2 mutant strain by nucleic acid amplification and melting curve analysis using a reagent combining probes of SEQ ID NO: 1 and SEQ ID NO: 2, a reagent combining probes of SEQ ID NO: 3 and SEQ ID NO: 2, a reagent combining probes of SEQ ID NO: 3 and SEQ ID NO: 4, and a reagent combining probes of SEQ ID NO: 29 and SEQ ID NO: 2. The results are summarized in Table 1 below.

[0067] [Table 1]

[0068] As shown in Figures 1 to 4, it was found that even if a probe combination that can confirm a good detection peak in the Delta strain is used, the detection peak waveform may be disturbed and the maximum fluorescence change rate may be significantly reduced in the Omicron strain, which is known to have many mutations. On the other hand, even when the same probe of SEQ ID NO: 2 is used, when SEQ ID NO: 3 or 29 is combined, it was confirmed that the detection peak waveform is not disturbed even in the Omicron strain and a high maximum fluorescence change rate is shown. In addition, when SEQ ID NO: 3 is used, it was confirmed that similarly good results can be obtained even when it is combined with a probe of SEQ ID NO: 4, which is different from SEQ ID NO: 2.

[0069] [Test Example 2: Evaluation of SARS-CoV-2 detection using a combination of nucleic acid probes] The following test was carried out to confirm whether the effect obtained by combining the probe of SEQ ID NO: 3 used in Test Example 1 could be obtained even when the probe of SEQ ID NO: 2 or 4 was replaced with another probe. Specifically, in this test, detection was carried out when each of the probes of SEQ ID NO: 2 and 4 to 11 was used alone and when the probe was combined with the probe of SEQ ID NO: 3, and the results were compared to examine whether there was a synergistic effect due to the combination. (1) Sample preparation The positive control RNA for SARS-CoV-2 (EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.)) was diluted with sterile water to 10 copies / test and used as the sample. (2) Reverse transcription, nucleic acid amplification, melting curve analysis, and evaluation The above samples were added to the following reagents, and SARS-CoV-2 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis.

[0070] (reagent) The probes of SEQ ID NO: 2 and 4 to 11 and the probe of SEQ ID NO: 3 were prepared, and the following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). For each probe, the probes of SEQ ID NO: 2 and 4 to 11 were used alone or in combination with the probe of SEQ ID NO: 3. When the probe of SEQ ID NO: 3 was used in combination, the primer set of SEQ ID NO: 25 and 26 was also used. Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were used in the amounts adjusted as described in the 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 was performed normally. (When the probes of SEQ ID NOs: 2 and 4 to 11 are used alone) 0.5 μM primer shown in SEQ ID NO: 25 3.0 μM primer shown in SEQ ID NO:26 0.25 μM of a probe represented by any one of SEQ ID NOs: 2 and 4 to 11 (5' or 3' end labeled with BODIPY-FL) (When probes of SEQ ID NO: 2 and SEQ ID NO: 4 to 11 are used in combination with SEQ ID NO: 3) 0.5 μM primer shown in SEQ ID NO: 23 3.0 μM primer shown in SEQ ID NO:24 0.25 μM of the probe shown in SEQ ID NO:3 (labeled at the 5' end with BODIPY-FL) 0.5 μM primer shown in SEQ ID NO: 25 3.0 μM primer shown in SEQ ID NO:26 0.25 μM of a probe represented by any one of SEQ ID NOs: 2 and 4 to 11 (5' or 3' end labeled with BODIPY-FL)

[0071] (Reverse transcription, nucleic acid amplification and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (One cycle) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0072] (3) Results The detection results when the probes of SEQ ID NO:2 and 4 to 11 were used alone are shown in Table 2 below, and the detection results when the probes of SEQ ID NO:2 and 4 to 11 were used in combination with the probe of SEQ ID NO:3 are shown in Table 3 below.

[0073] [Table 2]

[0074] [Table 3]

[0075] As shown in the results of Tables 2 and 3, there is no significant difference in the maximum fluorescence change rate when any of the probes of SEQ ID NO: 2 and 4 to 11 are used alone. However, when combined with the probe of SEQ ID NO: 3, the maximum fluorescence change rate hardly changes in the case of the probe of SEQ ID NO: 7, whereas the maximum fluorescence change rate increases significantly in the cases of the probes of SEQ ID NO: 2, 4 to 6, and 8 to 11. From these results, it became clear that in order to detect SARS-CoV-2 with high sensitivity and a high maximum fluorescence change rate, not just any combination of probes is acceptable, but it is important to select and use a specific combination of probes.

[0076] Figure 5 is a detection graph obtained when SARS-CoV-2 RNA was detected by nucleic acid amplification and melting curve analysis using a reagent combining the probes of SEQ ID NO: 4 and SEQ ID NO: 3. As shown in Figure 5, a single detection peak was confirmed at around 60 ° C by using a reagent combining these probes. Figure 6 is a detection graph obtained when SARS-CoV-2 RNA was detected by nucleic acid amplification and melting curve analysis using a reagent combining the probes of SEQ ID NO: 6 and SEQ ID NO: 3. Although the fluorescence value was slightly lower than that of the detection peak in Figure 5, it was confirmed that a detection peak could be confirmed even when the probes of SEQ ID NO: 6 and SEQ ID NO: 3 were combined.

[0077] [Test Example 3: Evaluation of SARS-CoV-2 detection using a combination of nucleic acid probes] The following test was carried out to confirm whether the effect of the probe combination confirmed in Test Examples 1 and 2 could be obtained even when the probe of SEQ ID NO: 3 was replaced with another probe. Specifically, in this test, detection was carried out when the probes of SEQ ID NO: 3, 12 to 22, and 29 were used alone and when these probes were combined with the probe of SEQ ID NO: 2, and the results were compared to examine whether there was a synergistic effect due to the combination. (1) Sample preparation The positive control RNA for SARS-CoV-2 (EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.)) was diluted with sterile water to 10 copies / test and used as the sample. (2) Reverse transcription, nucleic acid amplification, melting curve analysis, and evaluation The above samples were added to the following reagents, and SARS-CoV-2 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis.

[0078] (reagent) The probes of SEQ ID NO: 3 and 12 to 22 and the probe of SEQ ID NO: 2 were prepared, and the following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). For each probe, the probes of SEQ ID NO: 3, 12 to 22, and 29 were used alone or in combination with the probe of SEQ ID NO: 2. When the probe of SEQ ID NO: 2 was used in combination, the primer set of SEQ ID NO: 25 and SEQ ID NO: 26 was also used. Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were used in the amounts adjusted as described in the 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 was performed normally. (When the probe of SEQ ID NO: 3, SEQ ID NO: 12 to 22, or SEQ ID NO: 29 is used alone) 0.5 μM primer shown in SEQ ID NO: 23 3.0 μM primer shown in SEQ ID NO:24 0.25 μM of a probe represented by any one of SEQ ID NOs: 3 and 12 to 22 (5' or 3' end labeled with BODIPY-FL) (When the probe of SEQ ID NO: 3, SEQ ID NO: 12 to 22, or SEQ ID NO: 29 is used in combination with SEQ ID NO: 2) 0.5 μM primer shown in SEQ ID NO: 23 3.0 μM primer shown in SEQ ID NO:24 0.25 μM of a probe represented by any one of SEQ ID NOs: 3, 12 to 22, and 29 (5' or 3' end labeled with BODIPY-FL) 0.5 μM primer shown in SEQ ID NO: 25 3.0 μM primer shown in SEQ ID NO:26 0.25 μM probe shown in SEQ ID NO:2 (3' end labeled with BODIPY-FL)

[0079] (Reverse transcription, nucleic acid amplification and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (One cycle) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0080] (3) Results The detection results when the probes of sequence numbers 3, 12 to 22, and 29 were used alone are shown in Table 4 below, and the detection results when the probes of sequence numbers 3, 12 to 22, and 29 were used in combination with the probe of sequence number 2 are shown in Table 5 below.

[0081] [Table 4]

[0082] [Table 5]

[0083] As shown in the results of Tables 4 and 5, it was shown that, as in Test Examples 1 and 2, some combinations of two probes significantly improve the maximum fluorescence change rate. From these results, it was revealed that as a probe to be combined with the probe of SEQ ID NO: 2 or the probes of SEQ ID NO: 4 to 6 and 8 to 11, which have been confirmed to have the same effect as the probe of SEQ ID NO: 2, any of the probes of SEQ ID NO: 3, 13, 14, 16 to 22, and 29 is preferable, and among them, by using any of the probes of SEQ ID NO: 3, 14, 16 to 18, and 29 in combination, a particularly high maximum fluorescence change rate can be achieved, enabling more sensitive detection.

[0084] FIG. 7 is a detection graph obtained when SARS-CoV-2 RNA was detected by nucleic acid amplification and melting curve analysis using a reagent combining the probes of SEQ ID NO: 3 and SEQ ID NO: 2. As shown in FIG. 7, a single detection peak was confirmed at around 60°C by using a reagent combining these probes. FIG. 8 is a detection graph obtained when SARS-CoV-2 RNA was detected by nucleic acid amplification and melting curve analysis using a reagent combining the probes of SEQ ID NO: 12 and SEQ ID NO: 2. As shown in FIG. 8, it was observed that the detection peak waveform was distorted and the maximum fluorescence change rate value was reduced when using a reagent combining these probes.

[0085] [Test Example 4: Detection of SARS-CoV-2 positive clinical specimens by combination of nucleic acid probes] (1) Sample preparation In routine medical practice, 10 nasopharyngeal swabs (positive samples with unknown concentration) from subjects diagnosed with SARS-CoV-2 were used as samples. First, 10 μL of proteinase K solution (Kanto Chemical) was dispensed into a 1.5 mL tube, and the nasopharyngeal swabs from each subject were added and mixed. Next, these mixtures were heated at 65°C for 5 minutes, then heated at 95°C for another 5 minutes, and the reaction solution was cooled sufficiently. The reaction solution was mixed with an equal amount of GeneCube dedicated dissolving solution (Toyobo) to prepare the sample preparation solution. Note that this sample corresponds to a sample containing impurities derived from the sample, since it was not subjected to nucleic acid purification treatment. (2) Reverse transcription, nucleic acid amplification, melting curve analysis, and evaluation The above samples were added to the following reagents, and SARS-CoV-2 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis.

[0086] (reagent) A combination of SEQ ID NO: 1 and SEQ ID NO: 2, and a combination of SEQ ID NO: 29 and SEQ ID NO: 2 were prepared as probe sets, and the following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were used in the amounts adjusted as described 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 was performed normally. 0.5 μM primer shown in SEQ ID NO: 23 3.0 μM primer shown in SEQ ID NO:24 0.25 μM probe shown in SEQ ID NO: 1 or 29 (5' or 3' end labeled with BODIPY-FL) 0.5 μM primer shown in SEQ ID NO: 25 3.0 μM primer shown in SEQ ID NO:26 0.25 μM of the probe shown in SEQ ID NO:2 (labeled at the 3' end with BODIPY-FL)

[0087] (Reverse transcription, nucleic acid amplification and melting curve analysis) 42℃・2 minutes 97℃・15 seconds (One cycle) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0088] (3) Results As a representative example, a graph of the results of measuring sample No. 2 using a reagent combining the probes of SEQ ID NO: 29 and SEQ ID NO: 2 is shown in Figure 9. The results of detecting 10 SARS-CoV-2 positive samples by nucleic acid amplification and melting curve analysis using a reagent combining the probes of SEQ ID NO: 1 and SEQ ID NO: 2, and a reagent combining the probes of SEQ ID NO: 29 and SEQ ID NO: 2, are shown in Table 6.

[0089] [Table 6]

[0090] As shown in Table 6, when 10 positive samples with unknown concentrations were measured using the combination of SEQ ID NO: 1 and SEQ ID NO: 2, and the combination of SEQ ID NO: 29 and SEQ ID NO: 2, all were determined to be positive for the combination of SEQ ID NO: 29 and SEQ ID NO: 2, but one sample was determined to be negative for the combination of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, since infection with the Omicron strain is currently the mainstream, it was found that the waveform of the detection peak was distorted in all but one sample for the combination of SEQ ID NO: 1 and SEQ ID NO: 2. Furthermore, it was confirmed that the combination of SEQ ID NO: 29 and SEQ ID NO: 2 did not distort the waveform of the detection peak and had a higher maximum fluorescence intensity. [Industrial Applicability]

[0091] By using the nucleic acid probes of the present invention in combination, for example, in melting curve analysis, SARS-CoV-2 that may be contained in a sample can be detected easily, quickly, and accurately without differences between strains. The present invention makes it possible to detect SARS-CoV-2 with high reliability, which can greatly contribute to clinical diagnosis, etc.

Claims

1. A probe set including the following fluorescently labeled probe (A) and the following fluorescently labeled probe (B), used for detecting SARS-CoV-2 that may be contained in a specimen sample: (A) a fluorescently labeled probe comprising a base sequence A1 represented by any one of SEQ ID NOs: 3, 13, 14, 16 to 22, and 29, a base sequence A2 complementary to the base sequence A1, or a base sequence A3 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence A1 or A2, and labeled with a fluorescent dye; and (B) A fluorescently labeled probe comprising a base sequence B1 represented by any one of SEQ ID NOs: 2, 4 to 6, and 8 to 11, a base sequence B2 complementary to the base sequence B1, or a base sequence B3 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence B1 or B2, and which is labeled with a fluorescent dye.

2. The probe set according to claim 1 , wherein each of the fluorescently labeled probe (A) and the fluorescently labeled probe (B) is labeled at only one of its 5′ end and 3′ end.

3. 2. The probe set according to claim 1, wherein the fluorescent dye (A) is a fluorescent quenching dye that is quenched when the fluorescent labeled probe (A) is 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 fluorescent labeled probe, and the fluorescent dye (B) is a fluorescent quenching dye that is quenched when the fluorescent labeled probe (B) is 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 fluorescent labeled probe.

4. The probe set according to claim 1 , wherein the fluorescent dye (A) and the fluorescent dye (B) are fluorescence quenching dyes that are quenched by interaction with guanine.

5. The probe set according to claim 1, wherein the fluorescent dye (A) and the fluorescent dye (B) are at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

6. The probe set according to claim 1, wherein the fluorescent dye (A) and the fluorescent dye (B) are at least one fluorescent 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, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

7. The probe set according to claim 1 , wherein the labeled terminal base of both the fluorescently labeled probe (A) and the fluorescently labeled probe (B) is cytosine.

8. The probe set according to claim 1, which is used to detect SARS-CoV-2 that may be contained in a specimen sample derived from a living organism that has not been subjected to a nucleic acid purification process.

9. A method for detecting SARS-CoV-2 that may be contained in a specimen sample, using a probe set according to any one of claims 1 to 8.

10. The method according to claim 9, comprising the step of detecting by RT-PCR-melting curve analysis.

11. The method according to claim 10, wherein the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

12. 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.

13. The method according to claim 10, wherein the enzyme having reverse transcription activity used in the RT reaction is a reverse transcriptase derived from M-MLV (Moloney Murine Leukemia Virus) or a mutant thereof.

14. The method according to claim 9 , wherein the specimen sample is a specimen sample derived from a living organism that has not been subjected to a step of purifying nucleic acid.

15. A reagent kit for detecting SARS-CoV-2 that may be contained in a specimen sample, comprising the probe set according to any one of claims 1 to 8.

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