Oligonucleotides for detecting SARS-CoV-2 and their uses

Abstract

To provide a technique for detecting SARS-CoV-2.SOLUTION: As a labeled probe used for detecting SARS-CoV-2, which can be included in a sample, used is one having following characteristics (A) and (B): (A) it has a base sequence at least 85% identical to a base sequence represented by a specific sequence or another different sequence or a base sequence complementary thereto including at least ten, continuous bases; and (B) only one of 5' end or 3' end is labeled. Preferably, the two or more labeled probes should be used so that they coexist in a reaction liquid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for quickly, reliably and simply detecting SARS-CoV-2 in a specimen. The present invention further relates to a method for detecting SARS-CoV-2 in a specimen using the oligonucleotide, and a reagent / kit for use in the method. [Background technology]

[0002] SARS-CoV-2 is the causative virus of the novel coronavirus disease (COVID-19), which was first identified in Wuhan, Hubei Province, China in December 2019. There are now seven types of coronaviruses pathogenic to humans, four of which (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) account for 10-15% of common cold cases, most of which are mild. The other two viruses (SARS-CoV (2002) and MERS-CoV (2012)) cause SARS and MERS, with MERS continuing to occur sporadically in the Middle East. COVID-19, caused by SARS-CoV-2, has been a pandemic (global epidemic) since December 2019.

[0003] Symptoms of COVID-19 range from asymptomatic to upper respiratory tract infection, pneumonia, and acute respiratory distress syndrome (ARDS). Elderly people and those with underlying diseases are particularly at high risk of developing severe symptoms, and many deaths have been reported so far. On the other hand, it has been reported that about 80% of infected people experience mild symptoms and many recover. However, such asymptomatic or mildly infected people may not realize they are infected and may become a source of infection. In addition, the number of pathogens in respiratory samples is small compared to influenza (1 / 100 to 1 / 1,000), making testing and diagnosis difficult. For these reasons, there is a strong demand today for a test method that can detect SARS-CoV-2 easily, quickly, and with high sensitivity.

[0004] Genetic, antigen, and antibody tests have been developed so far as methods for testing SARS-CoV-2. Of these, antigen tests have the advantage of being easy to use, but they currently have problems in terms of 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 characteristic of being able to specifically detect SARS-CoV-2 with excellent sensitivity, and are currently the definitive method for diagnosing SARS-CoV-2.

[0005] In genetic testing, the SARS-CoV-2 genome RNA is first reverse transcribed (RT) into cDNA using reverse transcriptase, and then the nucleic acid is amplified and detected using PCR or LAMP (Loop-mediated Isothermal Amplification). These methods are called RT-PCR and RT-LAMP, respectively. Currently, RT-PCR detection is mainly performed by quantitative detection using real-time PCR with a double-labeled nucleic acid probe (also called Taqman probe or hydrolysis probe). This method allows the amount of virus to be quantified based on control RNA, but since photometry is required for each PCR cycle, detection takes about 1 hour even in the shortest case. On the other hand, the RT-LAMP method is an excellent method that can amplify targets isothermally in a relatively short time, but the amplified product is confirmed by checking the turbidity caused by magnesium pyrophosphate generated during the reaction, so it may be difficult to identify whether the amplified reaction product is derived from the target gene. Furthermore, the RT-LAMP method has the problem that it is difficult to determine if amplification is inhibited by the sample because it is not possible to incorporate an internal control into the reaction system to confirm whether nucleic acid amplification has been performed normally (e.g., Non-Patent Documents 1, 2, 3, and 4).

[0006] Furthermore, 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 fluorescently labeled nucleic acid probes has the advantage of being easily adapted to genetic testing using automated analyzers. However, there are still no known nucleic acid probes that can detect SARS-CoV-2 with high sensitivity using melting curve analysis.

[0007] However, RNA viruses such as SARS-CoV-2 are prone to frequent mutations, and when amplification and detection are performed using the RT-PCR method, if a mutation occurs in the sequence to which the primer or probe binds (anneals), it may not be possible to amplify or detect it. Therefore, by amplifying and simultaneously detecting multiple regions, even if PCR does not work in one region, it is possible to detect it if the other is amplified, and as a result, more reliable and sensitive detection is possible even for RNA viruses that are prone to frequent mutations. On the other hand, putting multiple primer-probe sets into the same reaction system increases the possibility of interactions such as forming dimers between oligonucleotides, and more trial and error is required to find the optimal primer and probe sequences.

[0008] In addition, in SARS-CoV-2 testing, RNA is usually extracted and purified from samples such as nasopharyngeal swabs, sputum, and saliva using an RNA extraction and purification kit, and the RNA extract is then used in the next step, such as RT-PCR. However, because RNA extraction and purification requires a great deal of effort and cost, there is a demand for samples prepared with only simple pretreatment to be used for testing. In other words, there is a demand for a method that allows detection using samples from which RNA has been simply extracted (simple RNA extraction solution), rather than extracting and purifying SARS-CoV-2 genomic RNA from samples containing SARS-CoV-2.

[0009] As described above, there is a demand for measurement methods for SARS-CoV-2 testing that have the following advantages: (1) short measurement time, (2) high sensitivity, (3) resistance to amplification inhibition by sample specimens, (4) ability to respond to SARS-CoV-2 mutations, and (5) ability to incorporate an internal control that can detect amplification inhibition by sample specimens into the reaction system. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Pathogen Detection Manual 2019-nCoV Ver.2.9.1 (March 19, 2020) National Institute of Infectious Diseases [Non-Patent Document 2] In Vitro Diagnostic Drug SARS Coronavirus Nucleic Acid Kit Cobas (registered trademark) SARS-CoV-2 Package Insert (1st edition created in April 2020), Roche Diagnostics K.K. [Non-Patent Document 3] SARS-CoV-2 Direct Detection RT-qPCR Kit Instruction Manual (revised May 13, 2020), Takara Bio Inc. [Non-Patent Document 4] In Vitro Diagnostic Drug SARS Coronavirus Nucleic Acid Kit Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit Package Insert (1st edition created in April 2020), Eiken Chemical Co., Ltd. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made against the background of the problems of the conventional technology. That is, the object of the present invention is to provide a method for detecting SARS-CoV-2 contained in a specimen, which has advantages such as being more rapid, highly sensitive, resistant to amplification inhibition by a simply extracted specimen, being able to respond to mutations of SARS-CoV-2, and being able to incorporate an internal control that can detect amplification inhibition by a specimen. [Means for solving the problem]

[0012] As a result of intensive research, the present inventors have discovered a method for detecting SARS-CoV-2 that can meet the above requirements by using a labeled probe having a specific sequence, particularly by using a combination of multiple specific labeled probes in a reaction solution, and have completed the present invention. That is, the outline of the present invention is as follows.

[0013] [Item 1] A labeled probe having the following characteristics (A) and (B) for use in detecting SARS-CoV-2 that may be contained in a sample: (A) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B) Only either the 5' or 3' end is labeled. [Item 2] The labeled probe according to Item 1, wherein the labeled probe comprises (A) a base sequence having 85% or more identity to the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto, the base sequence being at least 10 consecutive bases, Item 1. The labeled probe according to Item 1, which is characterized in that (A-2) it contains a base sequence of at least 10 consecutive bases in a base sequence showing 85% or more identity with the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto, and (B-2) it is used in the presence in a reaction solution of a labeled probe in which only one of the 5' end or the 3' end is labeled. [Item 3] The labeled probe according to Item 1, wherein the labeled probe comprises (A) a base sequence having at least 10 consecutive bases in a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto, Item 1. The labeled probe according to Item 1, characterized in that (A-1) it contains a base sequence of at least 10 consecutive bases in a base sequence showing 85% or more identity with the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto, and (B-1) it is used in the presence in a reaction solution together with a labeled probe in which only either the 5' end or the 3' end is labeled. [Item 4] The labeled probe according to any one of Items 1 to 3, wherein the base sequence of at least 10 bases described in (A) is a base sequence of at least 10 consecutive bases in the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted, or added in the base sequence. [Item 5] The labeled probe according to any one of Items 1 to 4, wherein the base sequence of at least 10 bases described in (A) consists of a base sequence shown in any one of SEQ ID NOs: 3 to 7 or 10 to 14, or a base sequence complementary thereto. [Item 6] The labeled probe according to any one of Items 1 to 5, wherein the label is a fluorescent dye label. [Item 7] The labeled probe according to any one of Items 1 to 6, wherein the labeled probe is labeled with a fluorescence quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the labeled probe. [Item 8] The labeled probe according to any one of Items 1 to 7, which is labeled with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 9] The labeled probe according to any one of Items 1 to 8, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 10] The labeled probe according to any one of Items 1 to 9, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 11] The labeled probe according to any one of Items 1 to 10, wherein in (B), the labeled terminal base is cytosine. [Item 12] A labeled probe according to any one of items 1 to 11, 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 13] A labeled probe set used to detect SARS-CoV-2 that may be contained in a sample, comprising at least the following first labeled probe and second labeled probe: (A-1) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-1) a first labeled probe, characterized in that only one of the 5' end or the 3' end is labeled; and (A-2) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-2) A second labeled probe, characterized in that only either the 5' end or the 3' end is labeled. [Item 14] A method for detecting SARS-CoV-2 that may be contained in a sample using a labeled probe according to any one of items 1 to 12 or a labeled probe set according to item 13. [Item 15] A method for detecting SARS-CoV-2 as described in Item 14, in which detection is performed by RT-PCR-melting curve analysis. [Item 16] The detection method according to Item 15, wherein the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to Family B. [Item 17] The detection method according to Item 16, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Item 18] The detection method according to any one of Items 14 to 17, 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 19] The detection method according to any one of Items 14 to 18, wherein the specimen sample is a biological sample that has not been subjected to a nucleic acid purification step. [Item 20] A reagent kit for detecting SARS-CoV-2 that may be contained in a sample, comprising the labeled probe described in any one of items 1 to 12 or the labeled probe set described in item 13. Effect of the Invention

[0014] According to the present invention, SARS-CoV-2 contained in a specimen can be detected quickly and with high sensitivity. According to the method of the present invention, even when a sample in which nucleic acid has not been highly isolated or purified, i.e., a sample from which RNA has been extracted simply, is used, it is difficult to be inhibited by amplification, and it is possible to detect SARS-CoV-2 with extremely high sensitivity. By using the detection method, reagent, or kit using the oligonucleotide of the present invention, SARS-CoV-2 can be detected easily, quickly, and with high sensitivity, which can greatly contribute to the field of clinical diagnosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in more detail below while showing embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. 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 addition, "and / or" in this specification means either one or both.

[0016] In addition, in this specification, a nucleic acid primer may be referred to as an oligonucleotide primer or simply as a primer, and a nucleic acid probe may be referred to as an oligonucleotide probe or simply as a probe, and these are collectively referred to as oligonucleotides.

[0017] [Methods for detecting SARS-CoV-2] One embodiment of the present invention is a method for detecting SARS-CoV-2 that may be contained in a specimen with high sensitivity. The present invention uses a labeled probe (also referred to as a "nucleic acid probe" in this specification) having a specific base sequence described below, preferably a combination of two or more types of the labeled probe, to rapidly, easily, and highly sensitively detect SARS-CoV-2 that may be contained in a specimen, for example, in an RT-PCR-melting curve analysis method. In a specific embodiment, the method of the present invention includes at least the following steps (1) to (3): (1) converting target RNA into cDNA in a specimen using an enzyme having reverse transcription activity; (2) performing a nucleic acid amplification reaction using one or more nucleic acid primer sets each having a specific base sequence, and generating one or more nucleic acid amplification products using the cDNA obtained in the step (1) as a template; and (3) detecting the one or more amplification products obtained in the step (2) by melting curve analysis using one or more labeled probes of the present invention consisting of specific base sequences; That is, one of the features of the present invention is that, in order to obtain highly sensitive determination results in detecting SARS-CoV-2, a labeled probe having one or more specific base sequences is used in a nucleic acid amplification detection reaction.

[0018] [Reverse transcription reaction] In one embodiment, the method of the present invention uses a nucleic acid primer and a reverse transcriptase to perform a reverse transcription (RT) reaction to generate cDNA from a target RNA. The reverse transcriptase is not particularly limited as long as it has reverse transcription activity, and examples thereof include reverse transcriptase (RNA-dependent DNA polymerase) derived from M-MLV (Moloney Murine Leukemia Virus) or AMV (Avian Myeloblastosis Virus) and its mutants. Examples of the mutants include mutants in which one or several amino acids are deleted, substituted and / or added in the wild-type amino acid sequence, and specifically, mutants in which RNase H activity is deleted 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 may be used as a reverse transcriptase in the present invention.

[0019] The nucleic acid primer used in the reverse transcription reaction may also serve as one of the PCR primers used in the subsequent nucleic acid amplification reaction. The conditions for the reverse transcription reaction are not particularly limited as long as the effects of the present invention are achieved, but for example, 37 to 55°C and 0 seconds to 60 minutes are preferred. Particularly preferred are 42 to 50°C and 2 to 15 minutes.

[0020] [Nucleic acid amplification reaction] In one embodiment, the method of the present invention uses a nucleic acid primer set to carry out a nucleic acid amplification reaction to generate a nucleic acid amplification product. The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level that allows visualization, 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 the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, TMA, etc. 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 carried out in the present invention is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[0021] [PCR reaction] PCR is a reaction catalyzed mainly by DNA polymerase, and the target nucleic acid is amplified by repeating three steps, namely (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase, as one cycle. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In the present invention, it is preferable to use a DNA polymerase belonging to family B from the viewpoints of rapidity, high sensitivity, and resistance to amplification inhibition by a sample. In addition, when performing 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.

[0022] The PCR reaction conditions are not particularly limited as long as the effects of the present invention are achieved, but for example, it is preferable to perform an initial heat deformation step at 80-100° C. for 0 seconds to 5 minutes, repeated heat deformation steps at 80-100° C. for 0.5-300 seconds, annealing at 35-80° C. for 1-300 seconds, and an extension reaction step at 35-85° C. for 1-300 seconds, and repeat this process 30-70 times. The temperature and time of the repeated cycles may be changed every one or several cycles.

[0023] [Family B DNA polymerase] The DNA polymerase used in the present invention 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, more preferably a DNA polymerase derived from bacteria of the genus Pyrococcus and Thermococcus. The present invention also includes a mutant of an archaea-derived DNA polymerase belonging to family B that does not lose its activity. The DNA polymerase mutant includes, but is not limited to, a mutant for the purpose of enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc. 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. 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. 9°N-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. PCR enzymes using these DNA polymerases are commercially available, and examples 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.

[0024] [DNA polymerase derived from KOD] In this 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, and / or adding one or several amino acids in the naturally-occurring amino acid sequence). In one preferred embodiment, the present invention performs 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, by using such a KOD DNA polymerase, it is possible to detect SARS-CoV-2 simply, quickly, and with high sensitivity, as shown in the examples described below.

[0025] [Nucleic acid primer set] The nucleic acid primer set described in (2) above used for detecting SARS-CoV-2 in the present invention is not particularly limited as long as it can amplify a nucleic acid fragment derived from SARS-CoV-2 that can form a complex with a labeled probe described below. From the viewpoint of easily obtaining a more sensitive determination result, the nucleic acid primer set is a nucleic acid primer set that can amplify a part or all of the nucleotide sequence of SARS-CoV-2 genomic RNA shown in SEQ ID NO: 15 or 16. For example, the nucleic acid primer set may be a nucleic acid primer set consisting of a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 2, or a nucleic acid primer consisting of a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 8 and a nucleic acid primer consisting of a nucleotide sequence shown in SEQ ID NO: 9, but is not limited thereto. For example, the nucleic acid primer set may be a combination of nucleic acid primers consisting of nucleotide sequences complementary to the nucleotide sequences of SEQ ID NOs: 1 and 2, respectively, or a combination of nucleic acid primers consisting of nucleotide sequences complementary to the nucleotide sequences of SEQ ID NOs: 8 and 9, respectively.

[0026] In this specification, SEQ ID NO: 15 and SEQ ID NO: 16 are nucleotide sequences corresponding to DNA obtained by reverse transcribing a part of the RNA sequence of SARS-CoV-2. The present invention is based on the new finding that the SARS-CoV-2 genomic RNA region corresponding to the nucleotide sequence shown in SEQ ID NO: 15 or 16 is used as a target sequence, and in particular, that the SARS-CoV-2 genomic region shown in SEQ ID NO: 15 and the SARS-CoV-2 genomic region shown in SEQ ID NO: 16 are simultaneously detected as target sequences, thereby enabling specific detection of SARS-CoV-2 with high sensitivity even in melting curve analysis, etc. Therefore, it is preferable that the nucleic acid primer set used in the present invention is capable of amplifying a part or all of both the regions shown in SEQ ID NO: 15 and 16.

[0027] [Step of detecting the amplification product] In the method of the present invention, step (3) includes a step of detecting the amplification products obtained in steps (1) and (2). The mode of this detection step is not particularly limited and can be carried out by any method known in the art, but in the present invention, melting curve analysis is preferred.

[0028] In one embodiment, the step (3) includes the following steps (3-1) to (3-2): (3-1) hybridizing the nucleic acid amplification product obtained in the step (2) with a nucleic acid probe to form a complex; and (3-2) A step of detecting the complex obtained in the step (3-1). In a preferred embodiment, the present invention is characterized in that a labeled probe capable of specifically reacting with a nucleic acid amplification product derived from SARS-CoV-2 obtained by steps (1) and (2) to form a complex is used in order to obtain a highly sensitive determination result in the detection of SARS-CoV-2. From the viewpoint of enabling highly sensitive detection even when only simple RNA extraction has been performed, it is particularly preferable to use one or more nucleic acid labeled probes capable of specifically reacting with one or more nucleic acid amplification products derived from SARS-CoV-2 obtained by steps (1) and (2) to form a complex.

[0029] [Nucleic acid probe] In one embodiment, the present invention is a nucleic acid probe that can specifically react with a nucleic acid amplification product derived from SARS-CoV-2 amplified with a nucleic acid primer set including the nucleic acid primers described above to form a complex. The labeled probe of the present invention that specifically reacts with a nucleic acid amplification product derived from SARS-CoV-2 can be a nucleic acid probe for detecting SARS-CoV-2 having the following characteristics: (A) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B) Only either the 5' or 3' end is labeled. By using a nucleic acid probe with such characteristics, it will be possible to detect SARS-CoV-2 with high sensitivity, for example, by melting curve analysis.

[0030] The nucleic acid probe of the present invention is not particularly limited as long as it has the base sequence of SEQ ID NO: 15 or SEQ ID NO: 16 or a sequence highly homologous to a base sequence complementary thereto. However, when the nucleic acid probe is labeled with a fluorescent quenching dye that is quenched by interaction with guanine as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0031] In one embodiment, the labeled probe used in the present invention may be a nucleic acid probe consisting of a base sequence showing 85% or more identity with the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, with at least 10 consecutive bases. Preferably, the nucleic acid probe consists of a base sequence of at least 10 consecutive bases in a base sequence showing 90% or more identity with the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, more preferably 93% or more identity, even more preferably 95% or more identity, even more preferably 98% or more identity. Particularly preferably, the nucleic acid probe consists of a base sequence 100% identical to the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a base sequence complementary thereto, with at least 10 consecutive bases.

[0032] In a specific embodiment, the labeled probe of the present invention may be, for example, a labeled probe consisting of a base sequence of at least 10 consecutive bases in a base sequence in which one or several bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16. Here, one or several may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2. In this way, by using a labeled probe having a base sequence highly homologous to the base sequence shown in SEQ ID NO: 15 or 16, it is possible to detect SARS-CoV-2 with high sensitivity.

[0033] In one preferred embodiment, the labeled probe of the present invention is a labeled probe consisting of a base sequence of at least 10 consecutive bases in the base sequence shown at positions 15 to 49 of the base sequence shown in SEQ ID NO: 15 or a base sequence complementary to said base sequence. By reacting such a labeled probe with a nucleic acid amplification product, it becomes possible to more effectively detect SARS-CoV-2.

[0034] In another preferred embodiment, the labeled probe of the present invention is a labeled probe consisting of a base sequence of at least 10 consecutive bases in the base sequence shown at positions 22 to 40 of the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto. By reacting such a labeled probe with the nucleic acid amplification product, it is possible to more effectively extract SARS-CoV-2.

[0035] The length of the labeled probe is not particularly limited as long as it is 10 bases or more, but may be, for example, 14 bases or more, preferably 14 to 25 bases, and more preferably 14 to 19 bases. By using a labeled probe of such a length, it is possible to more sensitively distinguish and detect SARS-CoV-2 with high sensitivity.

[0036] In a specific preferred embodiment, specific examples of the labeled probe used in the present invention include a labeled probe having a base sequence shown in any one of SEQ ID NOs: 3 to 7 or 10 to 14, or a base sequence complementary thereto. By using a labeled probe having such a specific base sequence, it becomes possible to detect SARS-CoV-2 with even higher sensitivity.

[0037] The above-mentioned labeled probe is characterized in that only one of the 5'-end or 3'-end is labeled. In one embodiment, the labeled probe of the present invention is preferably labeled to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 90% or more identity with a base sequence complementary to the base sequence of the labeled probe, and more preferably labeled to generate quenching. There is no particular limitation on the labeling substance, but it is more preferable to use a fluorescent dye label using a fluorescent substance.

[0038] The fluorescent label 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 is quenched by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that is quenched by interaction with guanine). 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).

[0039] 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, 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.

[0040] In certain preferred embodiments, a labeled probe in which the terminal base is cytosine and which is labeled with a fluorescent quenching dye is more preferred. When such oligonucleotide probes hybridize to amplification products, they can quench the light by forming base pairs with the guanine bases in the amplification products and interacting with them, making it very easy to measure changes in the fluorescence intensity of the reaction solution.

[0041] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the amplified 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 amplified 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).

[0042] In a further specific embodiment, even if at least one terminal base labeled with a fluorescence quenching dye (i.e., when both ends are labeled, at least one terminal base) is not cytosine in a nucleic acid probe, the change in 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 hybridizes, quenching can be achieved if a guanine base in the amplification product is present within a range of, for example, 1 to 3 bases relative to at least one terminal base labeled with a fluorescence quenching dye (a base that forms a base pair with the terminal base is counted as 1).

[0043] In a particular preferred embodiment, the method for detecting SARS-CoV-2 of the present invention uses two or more labeled probes of the present invention in combination in a detection reaction solution. By using a plurality of labeled probes of the present invention so that they coexist in the reaction solution, SARS-CoV-2 can be detected with even higher sensitivity. For example, by combining two or more labeled probes of the present invention so that they coexist in the detection reaction solution, it may be possible to detect SARS-CoV-2 with sufficient sensitivity even from a biological sample that has not undergone a process of purifying nucleic acids in advance.

[0044] In one preferred embodiment, (A) a labeled probe containing a base sequence of at least 10 consecutive bases in a base sequence showing 85% or more identity with the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto is used so as to coexist in a reaction solution with a labeled probe having the following characteristics (A-2) and (B-2): (A-2) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-2) Only either the 5' or 3' end is labeled.

[0045] In another preferred embodiment, (A) a labeled probe containing a base sequence of at least 10 consecutive bases in a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto is used so as to coexist in a reaction solution with a labeled probe having the following characteristics (A-1) and (B-1): (A-1) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-1) Only either the 5' or 3' end is labeled.

[0046] Therefore, the present invention further provides an embodiment of a labeled probe set comprising at least two of the above-mentioned labeled probes. The labeled probe set of the present invention can be used, for example, in a reaction solution in which a melting curve analysis method is performed, so that the multiple labeled probes contained in the set are coexistent. In one embodiment, the labeled probe set of the present invention is a labeled probe set used for detecting SARS-CoV-2 that may be contained in a specimen sample, comprising at least the following first labeled probe and second labeled probe: (A-1) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 15 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-1) a first labeled probe, characterized in that only one of the 5' end or the 3' end is labeled; and (A-2) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 16 or a base sequence complementary thereto, which contains a base sequence of at least 10 consecutive bases; and (B-2) A second labeled probe, characterized in that only either the 5' end or the 3' end is labeled. The base sequence, base length, fluorescent label, etc. of the labeled probe in the above-mentioned labeled probe set (also referred to as "nucleic acid probe set" in this specification) are the same as those described in detail for the above-mentioned labeled probe. The labeled probe set of the present invention can be used in the same manner as the above-mentioned labeled probe of the present invention.

[0047] In one preferred embodiment, in the above nucleic acid probe or nucleic acid probe set, the base sequence of at least 10 bases in (A-1) may be a base sequence shown in any one of SEQ ID NOs: 3 to 7 or a base sequence complementary thereto. In another preferred embodiment, in the above nucleic acid probe or nucleic acid probe set, the base sequence of at least 10 bases in (A-2) may be a base sequence shown in any one of SEQ ID NOs: 10 to 14 or a base sequence complementary thereto. In a particularly preferred embodiment, a nucleic acid probe or a nucleic acid probe set having the base sequences described in (A-1) and (A-2) above is used in combination.

[0048] [Step of detecting the complex] In the step of detecting the amplification product of the present invention, the embodiment of the step (3-2) of detecting a complex formed by hybridization of the nucleic acid amplification product with a labeled probe is not particularly limited, and may be carried out by any method known in the art.

[0049] In one embodiment, the step (3) may include at least one of the following steps (3-a) to (3-c): (3-a) A step of hybridizing the nucleic acid probe to the obtainable amplification product and monitoring the progress of the nucleic acid amplification reaction in real time by measuring the fluorescence intensity of the reaction solution. (3-b) After completion of the step (2), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the fluorescence intensity of the reaction solution to monitor the progress of the nucleic acid amplification reaction at an endpoint. (3-c) After completion of the step (2), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the temperature dependence of the fluorescence intensity of the reaction solution. According to the steps (3-a) to (3-c) described above, it is possible to easily and sensitively detect the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe. In the embodiment of the present invention, the detection step according to (3-b) or (3-c) is preferable from the viewpoint of more rapid detection of a nucleic acid amplification product. In particular, the method according to (3-c), i.e., melting curve analysis, is preferable.

[0050] [(3-a) Real-time monitoring of the progress of nucleic acid amplification reactions] This is a method for monitoring the progress of nucleic acid amplification reactions in real time. The so-called real-time PCR method allows for quantitative analysis by comparing with a control substance of known concentration. However, since photometry is required for each PCR cycle, the time required for nucleic acid amplification reactions tends to be long.

[0051] [(3-b) Monitoring the progress of nucleic acid amplification reaction at the endpoint] Although there are methods for monitoring the progress of a nucleic acid amplification reaction in real time, in the present invention, the progress of a nucleic acid amplification reaction is monitored at an end point, thereby enabling rapid detection of the target nucleic acid contained in a sample. Furthermore, the amount of the target nucleic acid can be roughly 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 an oligonucleotide probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured. The presence or absence of amplification of the target nucleic acid can be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of the reaction solution before the reaction. Alternatively, the presence or absence of the target nucleic acid contained in the 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. A control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the sample to be measured. The progress of a nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of making the amplification detection step quicker and easier, it is preferable to measure it at an end point.

[0052] [(3-c) Measuring the temperature dependence of fluorescence intensity] Measuring the temperature dependence of fluorescence intensity means, specifically, 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 oligonucleotide probe used can be obtained by first differentiating the obtained fluorescence intensity with respect to temperature. In addition, the fluorescence intensity may 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.

[0053] 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 oligonucleotide 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.

[0054] [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. For example, oral scrapings, pharyngeal swabs, nasal swabs, nasal aspirates, sputum, bronchial lavage, alveolar lavage, saliva, etc. collected from subjects suspected of infection with SARS-CoV-2 can be included, but are not limited to these. When a biological sample is used as the specimen sample to be measured, pretreatment such as dilution, suspension, centrifugation, enzyme treatment, or nucleic acid extraction may be performed depending on each biological sample, although this is not particularly limited.

[0055] The method of collecting and preparing the specimen is not particularly limited, and known methods can be used depending on the type and purpose of the specimen. In a particular preferred embodiment, the specimen used in the present invention does not need to be a sample from which RNA has been isolated and purified, and for example, a specimen collected from a living body may be treated with a protease (e.g., proteinase K) and / or heat treated (e.g., at 60 to 100°C for 1 second to 10 minutes) to degrade and denaturate the specimen, thereby degrading and removing RNase (ribonuclease) and DNase (DNA degrading enzyme) present in the specimen, and the resulting sample may be used as is.

[0056] 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 sample. As the nucleic acid, it may be a method for extracting and purifying mainly RNA, or a method for extracting and purifying nucleic acid without distinguishing between RNA and DNA. For example, a kit sold by each manufacturer may be used for nucleic acid extraction. For example, QIAamp Viral RNA Mini Kit (QIAGEN). In addition, a manual method or an automatic extraction and purification device may be used.

[0057] In a particular preferred embodiment, the method of the present invention can omit the nucleic acid purification step that is usually considered essential in conventional nucleic acid amplification reactions. As shown in the results of the test examples described below, the method of the present invention can detect SARS-CoV-2 with high sensitivity even in specimen samples containing contaminants such as biological components other than the target nucleic acid (which may be inhibitors of the nucleic acid amplification reaction). In addition to the need for dedicated reagents, nucleic acid purification has the problem of being complicated, time-consuming, and laborious. However, according to the gene amplification reaction of the present invention, there is no need to prepare a dedicated device for such nucleic acid purification, and it is possible to omit the complicated operation. Furthermore, since the time required for centrifugation can be shortened, the time from collection of a biological sample from a subject to obtaining a genetic test result can be shortened, and for example, the time from collection of a sample to obtaining a genetic test result can be within one day, preferably within half a day, more preferably within 6 hours, even more preferably within 3 hours, and especially preferably within 2 hours (e.g., within 1 hour). In this way, the labeled probe and method of the present invention, which can be used for detection using biological samples that have not undergone a nucleic acid purification process, eliminates the need for nucleic acid purification and enables simple and rapid detection of SARS-CoV-2.

[0058] [SARS-CoV-2] SARS-CoV-2 (also known as Severe Acute Respiratory Syndrome Coronavirus 2, a member of the Betacoronavirus genus) is the causative virus of the new coronavirus disease (COVID-19), which was first identified in Wuhan, Hubei Province, China in December 2019. There are now seven types of coronaviruses pathogenic to humans, four of which (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) account for 10-15% of common cold cases, most of which are mild. The other two viruses (SARS-CoV (2002) and MERS-CoV (2012)) cause SARS and MERS, with MERS continuing to occur sporadically in the Middle East. COVID-19, caused by SARS-CoV-2, has been a pandemic (global epidemic) since December 2019.

[0059] Symptoms of COVID-19 range from asymptomatic to upper respiratory tract infection, pneumonia, and acute respiratory distress syndrome (ARDS). In particular, elderly people and those with underlying diseases are at high risk of developing severe symptoms, and many deaths have been reported so far. On the other hand, it has been reported that about 80% of infected people have mild symptoms and many cases are cured. However, such asymptomatic or mildly infected people may not realize that they are infected and may become a source of infection. In addition, the number of pathogens in respiratory samples is small compared to influenza (1 / 100 to 1 / 1,000), making testing and diagnosis difficult. For these reasons, there is a strong demand today for a testing method that can detect SARS-CoV-2 easily, quickly, and with high sensitivity. In the present invention, it is possible to specifically detect SARS-CoV-2.

[0060] [Reagents for detecting SARS-CoV-2] Another embodiment of the present invention is a reagent for detecting SARS-CoV-2 that may be contained in a sample. The reagent contains at least components necessary for reverse transcription reaction (RT), nucleic acid amplification reaction, and detection, in addition to the nucleic acid probe or nucleic acid probe set of the present invention described above. The necessary components can be those known in the art. For example, it is preferable to contain at least a nucleic acid primer for reverse transcription, a nucleic acid primer set for PCR, reverse transcriptase, 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 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, the nucleic acid probe is preferably 0.01 to 1 μM, and more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, it is preferable that each nucleic acid probe contained in the probe set is within the above concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. The reverse transcriptase is preferably 0.01 to 10 U / μL, more preferably 0.02 to 2 U / μL. The DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.02 to 0.5 U / uL. The deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0061] Furthermore, additives known in the art may be added for the purpose of suppressing non-specific amplification or promoting the reaction. Known anti-DNA polymerase antibodies, phosphoric acid, etc. may be used as additives for suppressing non-specific amplification. Additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, and Nonidet P40. In addition, in the present invention, an inhibitor or suppressor of RNase may be added to reduce the degradation of the target genomic RNA of SARS-CoV-2. For example, an RNase inhibitor may be included. In the present invention, one or more of these additives may be used in combination, but is not limited to these.

[0062] [Kits for detecting SARS-CoV-2] Another embodiment of the present invention is a kit for detecting SARS-CoV-2 that may be contained in a sample. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe or nucleic acid probe set of the present invention described above, and / or a reagent containing these, and is configured to detect SARS-CoV-2. For example, the kit of the present invention can optionally contain a genetic testing reagent that can detect or quantify the presence of the target to be detected and / or an instruction manual explaining the method of use, etc. For example, the nucleic acid probes enclosed in the same container or in separate containers can be packaged in, for example, a single package and provided in a form including information on how to use the kit. In addition, a positive control solution or a negative control solution can be included. EXAMPLES

[0063] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0064] Example 1: Selection of nucleic acid probe N1 region (1) Sample preparation The positive control RNA for SARS-CoV-2 (EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.)) was serially diluted with sterile water to prepare samples with concentrations of 2.5, 5, and 10 copies per test. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis. Each concentration level was measured at n=8, and the detection rate was calculated by dividing the number of positives by 8.

[0065] reagent In order to evaluate the performance of each of the probes shown in SEQ ID NOs: 3 to 7, a solution containing the following reagents was prepared. KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM each of the probes shown in SEQ ID NOs: 3 to 7 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0066] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0067] (3) Results Table 1 below shows the detection rates when nucleic acid amplification and detection were performed under the above conditions. This study revealed that the nucleic acid probe shown in SEQ ID NO: 5 showed the highest detection rate among the nucleic acid probes shown in SEQ ID NO: 3 to 7.

[0068] [Table 1]

[0069] Example 2: Selection of nucleic acid probes N2 region (1) Sample preparation The positive control RNA for SARS-CoV-2 (EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.)) was serially diluted with sterile water to prepare samples with concentrations of 2.5, 5, and 10 copies per test. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis. Each concentration level was measured at n=8, and the detection rate was calculated by dividing the number of positives by 8.

[0070] reagent In order to evaluate the performance of each of the probes shown in SEQ ID NOs: 10 to 14, a solution containing the following reagents was prepared. KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM each of the probes shown in SEQ ID NOs: 10 to 14 (SEQ ID NOs: 10 and 13 are labeled with BODIPY-FL at the 3' end; SEQ ID NOs: 11 to 12 and 14 are labeled with BODIPY-FL at the 5' end) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0072] (3) Results Table 2 below shows the detection rates when nucleic acid amplification and detection were performed under the above conditions. This study revealed that the nucleic acid probe shown in SEQ ID NO: 10 showed the highest detection rate among the nucleic acid probes of SEQ ID NO: 10 to 14.

[0073] [Table 2]

[0074] Example 3: Examination of combinations of nucleic acid probes (1) Sample preparation The positive control RNA for SARS-CoV-2 (EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.)) was serially diluted with sterile water to prepare samples with concentrations of 2.5, 5, and 10 copies per test. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis. Each concentration level was measured at n=8, and the detection rate was calculated by dividing the number of positives by 8.

[0075] reagent In order to evaluate the coexistence of a primer probe set targeting the N1 region and a primer probe set targeting the N2 region in the same reaction system, a solution containing the following reagents was prepared by combining sequence numbers 5 or 3, and 10 or 13, which had high detection rates when used alone. KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM Probe shown by SEQ ID NO: 5 or 3 (labeled at the 3' end with BODIPY-FL) 0.125 μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM Probe shown in SEQ ID NO: 10 or 13 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0076] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0077] (3) Results Table 3 below summarizes the detection rates when nucleic acid amplification detection was performed under the above conditions. As shown in the results, it was confirmed that the detection rate of SARS-CoV-2, especially under low copy conditions, is improved by using multiple labeled probes of the present invention in combination. This study revealed that the highest detection rate and highest sensitivity were achieved when a primer probe set containing SEQ ID NO: 5 for detecting the N1 region was combined with a primer probe set containing SEQ ID NO: 10 for detecting the N2 region. Therefore, in the following examples, measurements were performed using this combination.

[0078] [Table 3]

[0079] Example 4: Measurement of minimum detection sensitivity of SARS-CoV-2 (1) Sample preparation Two types of positive control RNA for SARS-CoV-2 (AMPLIRUN SARS-CoV-2 RNA CINTROL (Vircell)) and EDX SARS-CoV-2 Standard (Bio-Rad Laboratories)) were serially diluted with sterile water to prepare samples with concentrations of 1, 2.5, 5, 10, 25, and 50 copies / test, respectively. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive when a peak was obtained in the melting curve analysis. Each concentration level was measured at n=8, and the detection rate was calculated by dividing the number of positives by 8.

[0080] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM Probe represented by SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL) 0.125 μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM Probe represented by SEQ ID NO: 10 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0081] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0082] (3) Results Table 4 below summarizes the results of nucleic acid amplification detection under the above conditions and detection of SARS-CoV-2 at low concentrations. The table shows the number of detections and the detection rate (the percentage obtained by dividing the number of positives by 8) in n=8 measurements. Table 4 reveals that the measurement method of the present invention can detect up to 2.5 copies / test in a pure system that does not use a sample.

[0083] [Table 4]

[0084] Example 5: Spiking of extracted samples using nasopharyngeal swabs (1) Sample preparation Nasopharyngeal swabs (negative specimens) from 16 cases were treated with the QIAamp Viral RNA Mini Kit (QIAGEN) to prepare RNA extracts. These were spiked with two types of SARS-CoV-2 positive control RNA (AMPLIRUN® SARS-CoV-2 RNA CONTROL (Vircell) and EDX SARS-CoV-2 Standard (Bio-Rad Laboratories)) at 10 copies / test to prepare the samples. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive if a peak was obtained in the melting curve analysis.

[0085] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM Probe represented by SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL) 0.125 μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM Probe represented by SEQ ID NO: 10 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0086] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0087] (3) Results Table 5 below summarizes the results of nucleic acid amplification detection under the above conditions and detection of SARS-CoV-2. In the measurement of 16 nasopharyngeal swabs (RNA extracts) spiked with two types of SARS-CoV-2 positive control RNA at 10 copies / test each, SARS-CoV-2 was detected in all 32 assays. This demonstrates that the measurement method of the present invention can stably detect 10 copies / test when purified RNA extracts are used as samples.

[0088] [Table 5]

[0089] [Example 6: Pseudovirus detection test (pure system)] (1) Sample preparation A pseudovirus incorporating SARS-CoV-2 genomic RNA (AccuPlex SARS-CoV-2 Reference Material Kit (SeraCare)) was diluted to 5 copies per test. The diluted solution was then treated with proteinase K (Kanto Chemical) and heat treatment to prepare a simple RNA extract, which was 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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive if a peak was obtained in the melting curve analysis.

[0090] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM Probe represented by SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL) 0.125 μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM Probe represented by SEQ ID NO: 10 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0091] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0092] (3) Results Table 6 below summarizes the results of nucleic acid amplification detection under the above conditions to detect SARS-CoV-2. All n=8 assays detected 5 copies / test of the pseudovirus. This demonstrates that the measurement method of the present invention is capable of detecting extremely low copy numbers.

[0093] [Table 6]

[0094] Example 7: Pseudovirus spike test (saliva sample) (1) Sample preparation A pseudovirus incorporating SARS-CoV-2 genomic RNA (AccuPlex SARS-CoV-2 Reference Material Kit (SeraCare)) was spiked into saliva samples (10 cases) to a concentration of 10 copies / test. A simple RNA extract was then prepared using proteinase K (Kanto Chemical) and heat treatment, and this was used as the sample. This sample was used as is without further nucleic acid isolation and purification, and therefore corresponds to a sample containing biological contaminants. (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. Toyobo GENECUBE (registered trademark) was used for reverse transcription, nucleic acid amplification, and melting curve analysis. The result was judged as positive if a peak was obtained in the melting curve analysis.

[0095] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer shown in SEQ ID NO: 1 0.25 μL 10 μM primer shown in SEQ ID NO: 2 1.5 μL 10 μM Probe represented by SEQ ID NO:5 (labeled at the 3' end with BODIPY-FL) 0.125 μL 10 μM primer represented by SEQ ID NO: 8 0.25 μL 10 μM primer shown in SEQ ID NO: 9 1.5 μL 10 μM Probe represented by SEQ ID NO: 10 (labeled at the 3' end with BODIPY-FL) 0.125 μL 5U / μL reverse transcriptase (Toyobo) 0.5μL Sample 4μL

[0096] 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 60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec)

[0097] (3) Results Table 7 below summarizes the results of nucleic acid amplification detection under the above conditions and detection of SARS-CoV-2. SARS-CoV-2 was detected in a total of 20 assays, measuring 10 simple RNA extracts from saliva samples spiked with 10 copies of pseudovirus per test (n=2). This revealed that the measurement method of the present invention can stably detect SARS-CoV-2 without amplification inhibition even in samples that have not been subjected to nucleic acid isolation and purification treatment and contain biological contaminants. In other words, it was revealed that the method has high resistance to amplification inhibition by nucleic acid amplification inhibitors originally used in the sample.

[0098] [Table 7] [Industrial Applicability]

[0099] By using the nucleic acid probe of the present invention, SARS-CoV-2 that may be present in a sample can be detected easily and with high sensitivity. Therefore, for example, the present invention enables rapid and reliable detection of samples containing low concentrations of SARS-CoV-2, which was previously difficult to detect, or samples from which simple RNA has been extracted, and is expected to make a significant contribution to clinical diagnosis, etc.

Claims

1. A labeled probe used to detect SARS-CoV-2 that may be contained in a specimen sample, the labeled probe having the following characteristics (A) and (B): (A) consisting of a base sequence shown in any one of SEQ ID NOs: 3, 5 to 7, 10 and 13, or a base sequence complementary thereto; and (B) Only either the 5' or 3' end is labeled.

2. The labeled probe of claim 1 , wherein the label is a fluorescent dye label.

3. 3. The labeled probe according to claim 1, which is labeled with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that shows 90% or more identity with a base sequence complementary to the base sequence of the labeled probe.

4. 4. The labeled probe according to claim 1, which is labeled with a fluorescence quenching dye that is quenched by interaction with guanine.

5. The labeled probe according to any one of claims 1 to 4, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof.

6. The labeled probe according to any one of claims 1 to 5, wherein the label of (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

7. The labeled probe according to any one of claims 1 to 6, wherein in (B), the labeled terminal base is cytosine.

8. The labeled probe according to any one of claims 1 to 7, which is used for detecting SARS-CoV-2 that may be contained in a specimen sample derived from a living body that has not been subjected to a nucleic acid purification step.

9. A labeled probe set used for detecting SARS-CoV-2 that may be contained in a specimen sample, comprising at least the following first labeled probe and second labeled probe: (A-1) consisting of a base sequence shown in any one of SEQ ID NOs: 3, 5 to 7 or a base sequence complementary thereto; and (B-1) a first labeled probe, characterized in that only one of the 5'-end or 3'-end is labeled; and (A-2) consisting of a base sequence shown in any one of SEQ ID NOs: 10 and 13 or a base sequence complementary thereto; and (B-2) A second labeled probe, characterized in that only either the 5' end or the 3' end is labeled.

10. A method for detecting SARS-CoV-2 that may be contained in a specimen sample, using the labeled probe according to any one of claims 1 to 8 or the labeled probe set according to claim 9, and the primer set according to SEQ ID NOs: 1 and 2 or SEQ ID NOs: 8 and 9.

11. The method for detecting SARS-CoV-2 according to claim 10, wherein the detection is performed by RT-PCR-melting curve analysis.

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

13. The detection method according to claim 12, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof.

14. The detection method according to any one of claims 10 to 13, 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.

15. The detection method according to any one of claims 10 to 14, wherein the specimen sample is a biological sample that has not been subjected to a step of purifying nucleic acid.

16. A reagent kit for detecting SARS-CoV-2 that may be contained in a specimen sample, comprising the labeled probe according to any one of claims 1 to 8 or the labeled probe set according to claim 9, and the primer set according to SEQ ID NOs: 1 and 2 or SEQ ID NOs: 8 and 9.

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