Improved virus detection method

Abstract

The present invention provides a method and a kit for making it possible to detect the presence / absence of virus RNA by one-step RT-PCR, in which the RNA is not isolated from a specimen, and a sample that has been subjected to only a simple thermal pre-treatment is added to a reaction solution. In particular, provided is a means for detecting, at a high sensitivity, the presence of a coronavirus in a specimen. A method for testing the presence of an RNA virus according to one embodiment of the present invention comprises steps (1) to (4): (1) a step for preparing a liquid mixture that contains a specimen which has not been subjected to RNA purification, an anionic polymer, and a polar organic solvent; (2) a step for heating the liquid mixture; (3) a step for adding, to the liquid mixture which has been heated, a 1-step RT-PCR reaction solution that includes (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; and (4) a step for performing 1-step RT-PCR reaction after tightly sealing the reaction container.

Description

Technical Field

[0001] The present invention relates to a method for detecting RNA viruses by nucleic acid amplification. More specifically, without isolating and purifying nucleic acids from a sample, after preparing a mixed solution containing the sample, an anionic polymer, and a polar organic solvent, heat treatment is performed, and the reaction solution of real-time reverse transcription polymerase chain reaction (qRT-PCR) is added, and the present invention relates to the detection of RNA viruses. According to the present invention, for example, it is possible to highly sensitively detect RNA viruses contained in biological samples such as saliva, throat swab fluid, nasal swab fluid, sputum, fecal samples, blood samples, environmental swab samples, etc. The present invention can be used in life science research, clinical diagnosis, food hygiene inspection, environmental inspection, etc.

Background Art

[0002] Viruses are roughly classified into DNA viruses having deoxyribonucleic acid as genomic nucleic acid and RNA viruses having ribonucleic acid. Since viruses have a short generation time, they are known to have a high mutation rate, and in particular, RNA viruses are said to be easily mutated. Such mutations in viruses are known to have a great influence on infectivity to hosts and the types and severities of symptoms when infected. Therefore, developing a method for quickly and accurately detecting even mutated viruses is important for preventing and containing the spread of infectious diseases.

[0003] Coronaviruses are the causative agents of respiratory infections, including the common cold, and it is estimated that approximately 10-35% of colds during outbreaks are caused by coronaviruses. Mutant viruses are known to occur, and in rare cases, deadly and severe respiratory illnesses such as SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and COVID-19 coronavirus (SARS-nCOV-2) have emerged. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronaviruses is crucial for clinical diagnosis, food hygiene testing, environmental testing, and other applications.

[0004] For pathogen testing of coronaviruses, methods such as electron microscopy, immunoassay-mediated antigen detection using ELISA, and detection of viral genes using nucleic acid amplification technology have been developed. Among these testing methods, nucleic acid amplification technology, which can detect coronaviruses with high sensitivity, is widely used. Several techniques have been developed to detect coronaviruses using nucleic acid amplification (for example, Non-Patent Document 1, Non-Patent Document 2, Patent Document 1).

[0005] In the case of the variant coronavirus SARS-nCOV-2, which was confirmed to have originated in Wuhan, Hubei Province, China in 2019, a testing method using nucleic acid amplification technology was established as soon as the analysis of the viral genome RNA was completed (e.g., Non-Patent Documents 3 and 4). In Japan, the National Institute of Infectious Diseases' "Pathogen Detection Manual 2019-nCoV" describes a method for detecting SARS-nCOV-2 (Non-Patent Document 5). In these methods, the detection of coronavirus contained in a sample involves the extraction and purification of viral RNA from the sample. The extraction and purification of viral RNA, especially the purification step, is complicated and requires a lot of work time. In recent years, a method has been known for detecting influenza viruses in which a viral extract obtained by mixing a pharyngeal swab sample with a pretreatment solution containing a water-soluble organic solvent and a surfactant is used as the sample (Patent Documents 2 and 3). In addition, K. Kang et al. have reported that highly pathogenic North American porcine genital respiratory syndrome virus RNA can be detected directly from porcine serum samples by RT-PCR (Non-Patent Document 6). These methods omit the RNA extraction and purification steps, which means that RT-PCR inhibitors contained in the sample are introduced into the reaction solution. The RT-PCR inhibitors vary greatly depending on the type of sample. For example, saliva samples introduce large amounts of PCR inhibitors such as polysaccharides and digestive enzymes like RNase. In addition, it is known that the conditions for viral inactivation and RNA extraction vary greatly depending on the virus species, but prior art literature makes no mention of the effect on coronaviruses (Patent Documents 2 and 3). Currently, there is a need for the development of a simple and rapid one-step RT-PCR method that can detect coronaviruses, especially SARS-nCoV-2 coronaviruses, from biological samples such as pharyngeal and nasal swabs, saliva, sputum, and fecal samples, as well as environmental swab samples, without the RNA purification step. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2012-24039 [Patent Document 2] Japanese Patent Publication No. 2017-023110 [Patent Document 3] Japanese Patent Publication No. 2016-182112 [Non-patent literature]

[0007] [Non-Patent Document 1] JOURNAL OF CLINICAL MICROBIOLOGY, Nov.2005,p.5452-5456 [Non-Patent Document 2] J Virol Methods. 2004 Sep 1;120(1):33-40. [Non-Patent Document 3] Published Online January29,2020,https: / / doi.org / 10.1016 / S0140-6736(20)30251-8 [Non-Patent Document 4] World Health Organization (WHO) website (Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR) [Non-Patent Document 5] National Institute of Infectious Diseases website, "Pathogen Detection Manual 2019-nCoV" (https: / / www.niid.go.jp / niid / images / lab-manual / 2019-nCoV20200217.pdf) [Non-Patent Document 6] J.Animal Science And Biotechnology, Volume 5, 2014, Page 45 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention has been made against the background of such problems of the prior art. That is, for example, from a sample containing a large amount of digestive enzymes and contaminants such as saliva, viral RNA, particularly RNA of a virus having an envelope, especially RNA of the coronavirus, without prior purification, by one-step RT-PCR, it is to enable simple, rapid, and highly sensitive detection.

Means for Solving the Problems

[0009] In view of the above circumstances, the present inventors conducted intensive research. As a result, in a sample in which viral RNA has not been purified, after mixing an anionic polymer and a polar organic solvent and then performing heat treatment, by subjecting it to one-step RT-PCR, it was found that RNA viruses (for example, coronaviruses, particularly SARS-nCOV-2) that may be contained in the sample can be detected, and the present invention was reached.

[0010] The representative invention of the present application is as follows. Item 1. A method for examining RNA viruses in a sample, characterized by including the following steps. (1) A step of preparing a mixed solution containing a sample in which RNA has not been purified, an anionic polymer, and a polar organic solvent. (2) A step of heating the mixed solution. (but) A step of adding a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity to the mixed solution after heating. (4) A step of performing a one-step RT-PCR reaction after sealing the reaction vessel. Item 2. The examination method according to Item 1, wherein in step (1), the content of the polar organic solvent in the mixed solution is 20% or more. Item 3. The inspection method according to Item 1 or 2, wherein the content of the anionic polymer in the mixed solution in step (1) is 0.00001% or more. Item 4. The inspection method according to any one of Items 1 to 3, wherein the mixed solution in step (1) substantially does not contain a surfactant. Item 5. The inspection method according to any one of Items 1 to 4, wherein the time from the preparation of the mixed solution in step (1) to the implementation of step (2) is within 5 minutes. Item 6. The inspection method according to any one of Items 1 to 5, wherein the heating condition in step (2) is 70°C for 1 second or more. Item 7. The inspection method according to any one of Items 1 to 6, wherein the sample is at least one selected from the group consisting of feces, throat swab, nasal swab, sputum, lung aspirate, cerebrospinal fluid, gargle, saliva, tear fluid, cultured cells, culture supernatant, and environmental swab test samples. Item 8. The inspection method according to any one of Items 1 to 7, wherein the sample is a suspension suspended in water, physiological saline, buffer solution or sputazyme enzyme solution, or their centrifuged supernatant or concentrate. <~ Item 9. The inspection method according to any one of Items 1 to 8, wherein the RNA virus is an RNA virus having an envelope. Item 10. The inspection method according to Item 9, wherein the RNA virus having an envelope is selected from the group consisting of Flaviviridae virus; Togaviridae virus; Coronaviridae virus; Orthomyxoviridae virus; Rhabdoviridae virus; Bunyaviridae virus; Paramyxoviridae virus; and Filoviridae virus. Item 11. The inspection method according to any one of Items 1 to 10, wherein the RNA virus having an envelope is a Coronaviridae virus. Item 12. The testing method described in Item 11, where the coronavirus family virus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCoV-2 coronavirus. Item 13. The testing method according to any one of items 1 to 12, characterized in that the RNA virus is an RNA virus that does not have an envelope. Item 14. The method for testing viruses according to Item 13, characterized in that the non-enveloped RNA virus is selected from the group consisting of astroviridae viruses; caliciviridae viruses; picornaviridae viruses; hepeviridae viruses; and reoviridae viruses. Item 15. The testing method according to any one of items 1 to 14, characterized in that the polar organic solvent is at least one selected from the group consisting of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pyridine, triethylamine dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, and acetonitrile. Item 16. The testing method according to any one of items 1 to 15, wherein the anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfate group, and a phosphonic acid group. Item 17. A method for testing for viruses according to any one of items 1 to 16, wherein the anionic polymer is at least one anionic polymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin, dermatan sulfate, polyvinyl sulfonic acid, polyvinylphosphonic acid, polystyrene sulfonic acid, polyacrylic acid, polyacrylic acid / sulfonic acid copolymer, polyacrylic acid / maleic acid copolymer and salts thereof. Item 18. The testing method according to any one of items 1 to 17, characterized in that the DNA polymerase is selected from the group consisting of Taq, Tth, and their variants. Item 19. The testing method according to any one of items 1 to 18, characterized in that the reverse transcriptase is selected from the group consisting of Moloney's mouse leukemia virus (MMRV), avian myeloblastosis virus (AMV), and their variants. The method for testing according to any one of items 1 to 19, characterized in that the one-step RT-PCR reaction solution in step (4) further comprises at least one selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum albumin, glycerol, glycol, and gelatin. Item 21. The method for testing for viruses as described in item 20, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. Item 22. A kit for testing RNA viruses, characterized by comprising an anionic polymer, a polar organic solvent, and a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcriptase activity. Item 23. A test kit according to item 22, further comprising at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin, glycerol, glycol, and gelatin. Item 24. A virus testing kit according to item 22 or 23, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected. Item 25. A virus testing kit according to any one of items 22 to 24, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected. Item 26. A virus testing kit according to any one of items 22 to 25, characterized in that the RNA virus has an envelope. Item 27. A virus testing kit according to Item 26, characterized in that the enveloped RNA virus is selected from the group consisting of Flaviviridae viruses; Togaviridae viruses; Coronavirusidae viruses; Orthomyxoviridae viruses; Rhabdoviridae viruses; Bunyaviridae viruses; Paramyxoviridae viruses; and Filoviridae viruses. Item 28. A test kit for the virus described in either item 26 or 27, wherein the enveloped RNA virus is a coronavirus. Item 29. A virus testing kit according to item 27 or 28, characterized in that the coronavirus family virus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2. Item 30. A virus testing kit according to any one of items 22 to 25, characterized in that the RNA virus does not have an envelope. Item 31. A virus testing kit according to Item 30, characterized in that the non-enveloped RNA virus is selected from the group consisting of astroviridae viruses; caliciviridae viruses; picornaviridae viruses; hepeviridae viruses; and reoviridae viruses. [Effects of the Invention]

[0011] This invention allows for the detection of RNA viruses in a sample without the need for nucleic acid isolation and purification. By mixing the sample with a reagent containing an anionic polymer and a polar organic solvent, followed by heat treatment, and then adding the mixture to a one-step RT-PCR reaction solution, the influence of contaminating substances such as RNases that may be present in unpurified samples can be significantly reduced. As a result, testing operations become more efficient, allowing for an increase in the number of tests performed and contributing to infectious disease prevention. Furthermore, the omission of the viral RNA purification step simplifies the process, reducing the risk of contamination between samples. This also reduces the risk of false positives and further improves the accuracy of testing operations. In addition, the reduction in the number of steps in which workers handle infectious samples reduces the risk of infection to workers.

[0012] Furthermore, the present invention can similarly demonstrate excellent effects even with samples that may contain the SARS-nCOV-2 coronavirus that emerged in 2019. In addition, the present invention enables highly sensitive detection of coronaviruses from samples containing many contaminants, such as biological samples including blood, feces (excreted stool, rectal stool), vomit, urine, sputum, lymph fluid, plasma, ejaculate, lung aspirate, cerebrospinal fluid, pharyngeal swabs, nasal swabs, gargle fluid, saliva, and tears, as well as environmental swab samples and samples containing cultured cells or culture supernatants. The present invention can be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, and the like. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the results of detecting viral RNA using a polar organic solvent containing an anionic polymer in the presence of RNase. [Figure 2] This figure shows the results of detecting inactivated viruses using polar organic solvents containing anionic polymers in the presence of RNase. [Figure 3] This figure shows the results of detecting viral RNA using a polar organic solvent containing an anionic polymer in the presence of a saliva sample. [Modes for carrying out the invention]

[0014] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited thereto. It should be understood that terms used herein are used in the sense commonly used in the art unless otherwise specified. Furthermore, all non-patent and patent documents mentioned herein are incorporated herein by reference. In this specification, "~" means "greater than or equal to, less than or equal to," for example, "X~Y" in the specification means "greater than or equal to X, less than or equal to Y." Also, "and / or" in this specification means either one or both. Furthermore, in this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified.

[0015] One aspect of the present invention is a method for testing for RNA viruses in a sample, comprising preparing a mixture containing the sample, an anionic polymer, and a polar organic solvent without purifying the viral RNA from the sample, then heat-treating the mixture, and adding a one-step RT-PCR reagent containing reverse transcriptase and DNA polymerase, or DNA polymerase having reverse transcription activity, for testing for the presence or absence of RNA viruses.

[0016] In a preferred embodiment, the method for testing a virus in a sample according to the present invention is characterized by comprising at least the following steps: (1) A step of preparing a mixture containing an RNA sample that has not been purified, an anionic polymer, and a polar organic solvent. (2) The step of heating the mixture. (3) Adding to the heat-treated mixture a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity, (4) After sealing the reaction vessel, the one-step RT-PCR reaction is carried out. It is preferable that steps (1) to (4) be carried out in the same container. That is, it is preferable not to transfer all or part of the mixture to another container between steps (1) to (4). The entire amount of the mixture from steps (1) and (2) may be used in steps (3) and (4), or a portion of it may be transferred to another container before carrying out steps (3) and (4).

[0017] In the present invention, the RNA virus to be tested may be an RNA virus having an envelope derived from a lipid bilayer, or an RNA virus without an envelope. In certain preferred embodiments, the present invention is particularly effective in enabling highly sensitive testing of enveloped RNA viruses from unpurified samples. Examples of enveloped RNA viruses (also called "enveloped RNA viruses") include, but are not limited to, Flaviviridae viruses (e.g., hepatitis C virus, Japanese encephalitis virus, Zika virus, swine fever virus); Togaviridae viruses (e.g., rubella virus, chikungunya virus); Coronaviruses (e.g., SARS coronavirus, MERS coronavirus, SARS-nCoV-2 coronavirus); Orthomyxoviridae viruses (e.g., influenza virus); Rhabdoviridae viruses (e.g., rabies virus); Bunyaviridae viruses (e.g., Crimean-Congo fever virus, hantavirus); Paramyxoviridae viruses (e.g., measles virus, human respiratory syncytial virus); Filoviridae viruses (e.g., Ebola virus). From the viewpoint of more reliably obtaining the effects of the present invention, it is preferably useful for detecting coronaviruses, more preferably useful for detecting SARS coronavirus, MERS coronavirus, and SARS-nCOV-2 coronavirus, and in particular useful for detecting SARS-nCOV-2 coronavirus (also called SARS-CoV-2).

[0018] The present invention can also be used for testing non-enveloped RNA viruses (also called "non-enveloped RNA viruses"). Examples of such non-enveloped RNA viruses include, but are not limited to, Astroviridae viruses (e.g., astrovirus); Caliciviridae viruses (e.g., sapovirus, norovirus); Picornaviridae viruses (e.g., hepatitis A virus, echovirus, enterovirus, coxsackievirus, poliovirus, rhinovirus); Hepeviridae viruses (e.g., hepatitis E virus); and Reoviridae viruses (e.g., rotavirus). However, it is preferably useful for detecting Caliciviridae viruses and Reoviridae viruses, more preferably for detecting norovirus, sapovirus, and rotavirus, and even more preferably for detecting norovirus and rotavirus, and particularly useful for detecting norovirus.

[0019] Examples of samples used in the present invention include pharyngeal swabs, nasal swabs, sputum, feces (excreted stool, rectal stool), vomit, and saliva, but are not particularly limited and can be used for any sample of biological origin. In particular, it is useful for detection from feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, gargle fluid, saliva, tears, cultured cells, and culture supernatants. These samples are characterized by containing large amounts of digestive enzymes such as proteases and nucleases (RNase, DNase) as impurities, and feces also contain large amounts of PCR reaction inhibitors such as proteins and nucleic acids derived from E. coli. It is known that reaction solution components such as enzymes, primers, and nucleic acid probes used in RT-PCR reactions are digested or inactivated by impurities contained in the sample, resulting in a decrease in detection sensitivity. Furthermore, nucleases contained in the sample may digest nucleic acids exposed from the virus, potentially reducing sensitivity or leading to false negative results due to undetectable results. In this invention, instead of isolating and purifying RNA using a commercially available RNA purification kit, the RNA is exposed from the viral structure by pre-heat treatment in a mixture containing an anionic polymer and a polar organic solvent, and then used in the RT-PCR reaction. The sample may be used directly for detection, or it may be a sample suspended in water, physiological saline, or buffer solution to reduce the influence of contaminants on the reaction and obtain more stable test results. In addition, for samples with particularly many contaminants, such as feces, centrifugation may be performed and the supernatant may be used. Alternatively, filter filtration may be performed. The buffer solution is not particularly limited, but examples include Hanks buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and Trisine buffer. Furthermore, in the case of highly viscous biological samples (for example, samples containing highly viscous sputum), although not particularly limited, samples treated with Suptazyme enzyme solution may also be used.

[0020] Another embodiment of the present invention involves a sample containing cultured cells or culture supernatant. Cell-based isolation culture is effective for virus isolation. Since the culture supernatant and cultured cells after isolation culture contain viruses, they can serve as samples in the present invention. Examples of cell types used for isolation culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells, and Sf21 cells. While the present invention is effective for samples containing U937 cells, which are known to contain a large amount of nucleases in the cell lysate obtained by thawing or disrupting cells, it is not particularly limited and encompasses a wide range of similar methods.

[0021] Another type of sample in this invention is a swab test sample from the environment. Swab tests are useful for elucidating contamination routes and understanding the contamination status of facility environments, etc. In this invention, a swab test is not particularly limited, but for example, it is a sample obtained by wiping the relevant area or equipment with a cotton swab, eluting it in water or a buffer solution, and concentrating it by polyethylene glycol (PEG) precipitation, etc. As an example of the procedure for a swab test, "Improvement of the Norovirus Testing Method for Swab Samples" (http: / / idsc.nih.go.jp / iasr / 32 / 382 / dj3824.html) is given, but it is not particularly limited, and a wide range of similar methods are included. Examples of areas to wipe include cooking utensils such as cutting boards, knives, dishcloths, and tableware, refrigerator handles, toilet and bathroom doorknobs, faucets in washrooms, kitchens, toilets, and bathrooms, the hands and fingers of cooks, bathrooms, toilets, washrooms, handrails, living rooms, and other facilities. Furthermore, although it is not a swab test, it can also be applied to concentrated sewage samples as an environmental test.

[0022] In this invention, polarity refers to the electronic imbalance within a molecule, and a molecule in which the centers of gravity of the positive and negative charges within the molecule do not coincide is called a polar molecule. A solvent composed of polar molecules is called a polar solvent. Among polar solvents, by using a polar organic solvent composed of organic compounds, it is possible to destabilize the higher-order structure of biomolecules such as nucleic acids and proteins. By utilizing this property, it is possible to weaken hydrophobic bonds in the structural proteins of viruses and destabilize the capsid structure. As mentioned above, the destabilizing effect of polar organic solvents on the capsid structure of viruses is known to differ depending on the type of virus. This is thought to be because the strength of hydrophobic bonds differs depending on the properties of the capsid protein and envelope possessed by the virus.

[0023] Examples of the aforementioned polar organic solvents include, but are not limited to, ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, acetonitrile, ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, pyridine, etc. Preferably, methanol, triethylamine, dimethyl sulfoxide, or acetone. Alternatively, a mixed solution containing two or more of these polar organic solvents may be used. The lower limit of the concentration of the polar organic solvent as a denaturant for capsid proteins depends on the type of polar organic solvent and other additives, but is not particularly limited as long as it is a concentration that denatures the capsid proteins. Furthermore, since the capsid proteins and envelopes differ depending on the type of virus, the effective concentration of the polar organic solvent differs for each virus, but usually the effective concentration of the polar organic solvent relative to the sample volume is 10% or more and less than 100%, more preferably 30% or more and 90%, and even more preferably 50% or more and 85%. For example, the effective concentration of the polar organic solvent can be achieved by making the content of the polar organic solvent in the mixed solution in step (1) 20% or more, preferably 25% or more, and more preferably 30% or more. The upper limit of the content of the polar organic solvent in the mixed solution in step (1) is not particularly limited as long as the effects of the present invention are achieved, but it is preferable to make it 90% or less, preferably 80% or less, and more preferably 75% or less. When two or more polar organic solvents are used in combination in the present invention, it is preferable to adjust them so that their total amount is within the range of the above content.

[0024] The aforementioned polar organic solvent is also commonly known as a PCR inhibitor. Therefore, by selecting a polar organic solvent in which the difference between the concentration required for protein denaturation and the permissible concentration for introduction into PCR is small, the detection operation can be easily carried out in the same container from capsid protein denaturation to the one-step RT-PCR reaction without opening and closing the container midway through, by sequentially adding the polar organic solvent, sample, and one-step RT-PCR reaction solution. Dimethyl sulfoxide is a particularly preferred example of such a polar organic solvent. For example, if 1 μL of dimethyl sulfoxide and 1 μL of sample are mixed in step (1), and 48 μL of one-step RT-PCR solution is added in step (3), the concentration of dimethyl sulfoxide introduced into the reaction solution is 2%. 2% dimethyl sulfoxide is a concentration that can be tolerated even if introduced into the RT-PCR solution. The amount of polar organic solvent (dimethyl sulfoxide) that can be suitably used in the present invention can be easily calculated in the same manner as described above.

[0025] The polar organic solvent may be used in combination with one or more surfactants, reducing agents, chelating agents, and metal salts, or it may be substantially free of these surfactants, etc. In certain embodiments, for example, the mixture in step (1) may be substantially free of surfactants. According to the present invention, even if pretreatment is performed in a state substantially free of surfactants, it is possible to detect RNA viruses in the sample with high sensitivity. Furthermore, since surfactants may inhibit the RT-PCR reaction depending on their type, it is desirable that the mixture be substantially free of surfactants. Here, substantially free means that the surfactant is not present in a concentration that allows nucleic acid extraction from RNA viruses. For example, the concentration of surfactant in the mixture in step (1) is 0.001% or less, preferably 0.0001% or less, more preferably 0.00001% or less, and among these, it is preferable that the mixture in step (1) contains no surfactants at all.

[0026] Anionic polymers are polymers formed by polymerization mainly of anionic monomers. For example, the anionic polymer used in the present invention is a polymer obtained by mainly polymerizing monomers having at least one anionic functional group selected from the group consisting of sulfonic acid groups, carboxyl groups, phosphate groups, sulfate groups, and phosphonic acid groups, and preferably a polymer obtained by polymerization using sulfonic acid groups as monomers. Nucleic acid molecules, including RNA and DNA, are also anionic polymers. Nucleic acid-degrading enzymes contained in the sample bind to nucleic acid molecules, which are anionic polymers, and digest them. Although we do not wish to be bound by theory, in the present invention, it is expected that adding an anionic polymer that is not digested by nucleic acid-degrading enzymes to the reaction solution system will have the effect of suppressing the digestion of target nucleic acid molecules.

[0027] The anionic polymer is not particularly limited as long as it achieves the effects of the present invention, but typical examples include nucleic acid polymers (polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid), polysaccharides (carrageenan, heparin, chondroitin sulfate, keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin, dermatan sulfate), polyvinyl sulfonic acid, polyvinylphosphonic acid, polystyrene sulfonic acid, polyacrylic acid, polyacrylic acid / sulfonic acid copolymer, polyacrylic acid / maleic acid copolymer, and the like.

[0028] The anionic polymer may be in the form of a salt. For example, it may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, etc.), or a hydrate salt. Preferably, it is an alkali metal salt, more preferably a sodium salt or potassium salt, and even more preferably a sodium salt.

[0029] The average molecular weight of the anionic polymer is not particularly limited as long as it achieves the effects of the present invention. In this specification, average molecular weight refers to weight-average molecular weight. The average molecular weight of the anionic polymer depends on the molecular weight and degree of polymerization of the constituent monomers, but may be, for example, 1,000 or more, preferably 5,000 or more, more preferably 10,000 or more, and even more preferably 50,000 or more. Furthermore, the upper limit of the average molecular weight of the anionic polymer is not particularly limited as long as it achieves the effects of the present invention, but may be, for example, 5,000,000 or less, preferably 1,000,000 or less, and more preferably 5,000,000 or less.

[0030] In certain embodiments, the content of the anionic polymer in the mixture of step (1) is preferably 0.00001% (v / v%) or more, more preferably 0.0001% or more, and even more preferably 0.001% or more. The upper limit of the content of the anionic polymer in step (1) is not particularly limited as long as the effects of the present invention are achieved, but for example, it can be 0.5% or less, more preferably 0.1% or less, and even more preferably 0.01% or less. By including the anionic polymer at such a concentration, it is possible to effectively prevent the digestion of RNA exposed from the virus in a sample that has not undergone the purification process, and / or to effectively prevent the influence of PCR inhibitors such as contaminants contained in the sample, and as a result, it is possible to highly suppress the decrease in the detection sensitivity of viral RNA. The content of the anionic polymer introduced into the one-step RT-PCR reaction solution in step (3) is not particularly limited as long as it does not inhibit the RT-PCR reaction, but may be, for example, 0.05% or less, and more specifically, 0.01% or less, or for example, 0.00001 to 0.001%.

[0031] In one embodiment, the RNA virus testing method of the present invention preferably has a time of 5 minutes or less between the preparation of the mixture in step (1) and the implementation of step (2). By shortening the time between the completion of step (1) and the start of step (2), faster RNA virus testing becomes possible. The time between the preparation of the mixture in step (1) and the implementation of step (2) is not particularly limited, but is preferably 5 minutes or less, more preferably 4 minutes or less, more preferably 3 minutes or less, even more preferably 2 minutes or less, and may be 1 minute or less. The lower limit of the time between the preparation of the mixture in step (1) and the implementation of step (2) is not particularly limited, but can be, for example, 10 seconds or more, preferably 30 seconds or more. In the present invention, even if the pretreatment time in step (1) is short, it is possible to induce the exposure of RNA contained in the envelope and capsid, and RNA detection by one-step RT-PCR becomes possible.

[0032] In one embodiment, the heating conditions in step (2) of the RNA virus testing method of the present invention may be 70°C or higher. Preferably, the effects of the present invention can be obtained more effectively by performing the test at 80°C or higher, more preferably at 90°C or higher, for example, 95°C. The upper limit of the heating conditions in step (2) is not particularly limited, but may be, for example, 100°C. Furthermore, it is preferable that the heating conditions in step (2) be at the above heating temperature for 1 second or more. In a particular preferred embodiment, it is preferable to heat for 30 seconds or more, 1 minute or more, or 3 minutes or more. The upper limit is not particularly limited as long as the effects of the present invention are achieved, but for example, by setting it to 10 minutes or less, rapid RNA virus testing becomes possible. For example, in one preferred embodiment, the heating conditions in step (2) may be 70°C for 1 second or more, or in another preferred embodiment, the heating conditions may be 80°C or 90°C for 1 second or more.

[0033] The process of purifying viral RNA from samples is complex and increases working time. In addition, transferring reaction vessels containing virus-containing samples and centrifugation pose a risk of virus and viral RNA dispersion. Virus dispersion threatens the safety and health of workers and also contaminates the testing environment. Since dispersed RNA viruses aerosolize in the workplace, there is a risk of contamination of other samples being tested simultaneously and infection of workers. Therefore, a method of testing for the presence or absence of virus using RT-PCR, which does not require the purification of viral RNA from samples, has significance beyond simply simplifying the process.

[0034] The one-step RT-PCR solution added to the aforementioned mixture contains reverse transcriptase and DNA polymerase. It is preferable to use a DNA polymerase that also possesses reverse transcriptase activity, such as Tth DNA polymerase or Taq DNA polymerase. More preferably, two enzymes are used, or at least two enzymes, such as reverse transcriptase and DNA polymerase.

[0035] The origin of the reverse transcriptase contained in the one-step RT-PCR reaction solution is not particularly limited as long as it can convert RNA to DNA, but examples include MMLV (Moloney Murine Leukemia Virus)-RT, AMV-RT (Avian Myeloblastosis Virus), HIV-RT, RAV2-RT, EIAV-RT, carboxydothermus hydrogenoformam DNA polymerase, and their variants. Particularly preferred examples include MMLV-RT, AMV-RT, or their variants.

[0036] The DNA polymerases included in the one-step RT-PCR reaction solution include, but are not limited to, Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. More preferably, Taq, Tth, or their variants are used. Particularly preferred is the use of Tth or its variants. Furthermore, to enhance the effect of suppressing nonspecific reactions, it is preferable to use it in combination with an anti-DNA polymerase antibody, or to introduce a thermally unstable block group into the DNA polymerase by chemical modification, thereby suppressing the enzymatic activity of the DNA polymerase during the reverse transcription reaction and enabling application to hot-start PCR.

[0037] In this specification, a DNA polymerase variant is defined as a variant that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more of the amino acid sequence identity with respect to the wild-type DNA polymerase from which it is derived, and that has the same DNA amplification activity as wild-type DNA polymerase, and, if necessary, the activity to convert RNA to cDNA. Here, the method for calculating amino acid sequence identity can be any means known in the art. For example, it can be calculated using commercially available analysis tools or tools available via telecommunication lines (the Internet). As an example, it is possible to calculate amino acid sequence identity by using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the mutants that can be used in the present invention are polypeptides consisting of amino acid sequences in which one or more amino acids are substituted, deleted, inserted, and / or added (hereinafter collectively referred to as "mutations") in the amino acid sequence of the wild-type DNA polymerase from which they are derived, and which may have the same activity as wild-type DNA polymerase in converting RNA to cDNA and amplifying DNA. Here, one or more may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3, but are not particularly limited.

[0038] The one-step RT-PCR reaction solution used in the present invention includes, in addition to reverse transcriptase and DNA polymerase, a buffer, a suitable salt such as a magnesium salt or manganese salt, deoxynucleotide triphosphate, and a primer pair corresponding to the target region of the viral RNA to be detected, and may further contain additives as needed.

[0039] The buffer used in this invention is not particularly limited, but examples include Tris, Tricin, Bis-Tricine, and Bicine. The pH is adjusted to 6-9, more preferably 7-8, with sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid. The concentration of the buffer added is 10-200 mM, more preferably 20-150 mM. At this time, a salt solution is added to create ionic conditions suitable for the reaction. Examples of salt solutions include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and ammonium acetate.

[0040] The dNTPs used in this invention are dATP, dCTP, dGTP, and dTTP, each added in concentrations of 0.1 to 0.5 mM, most commonly around 0.2 mM. A precaution against cross-contamination may be taken by using dUTP as a substitute for and / or in part for dTTP. Examples of magnesium salts include magnesium chloride, magnesium sulfate, and magnesium acetate, and examples of manganese salts include manganese chloride, manganese sulfate, and manganese acetate, with concentrations of 1 to 10 mM being preferred.

[0041] Furthermore, it is preferable that the additive included in the one-step RT-PCR reaction solution contains at least one selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum albumin, glycerol, glycol, and gelatin.

[0042] Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine, but it is not particularly limited to any quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid. The structure of the betaine-like quaternary ammonium salt is a compound that has both stable positive and negative charges within the molecule, exhibiting surfactant-like properties and is thought to cause destabilization of the viral structure. Furthermore, it is known to promote nucleic acid amplification by DNA polymerase. The preferred concentration of the betaine-like quaternary ammonium salt is 0.1 M to 2 M, and more preferably 0.2 M to 1.2 M.

[0043] The bovine serum albumin contained in the one-step RT-PCR reaction solution is preferably at least 0.5 mg / ml, more preferably at least 1 mg / ml. In samples with many impurities, a bovine serum albumin concentration of preferably 2 mg / ml or higher, and more preferably 3 mg / ml or higher, enables good detection.

[0044] The gelatin contained in the one-step RT-PCR reaction solution is derived from the skin, bones, tendons of animals such as cows and pigs, or from the scales and skin of fish, and is thought to contribute to the stabilization of the PCR enzyme. The concentration used should preferably be such that it stabilizes PCR amplification without interfering with fluorescence detection. Preferably, it is 1-5%, and more preferably 1-2%. There are no particular limitations on the origin of the gelatin, but fish-derived gelatin is preferred over cow- or pig-derived gelatin because it has lower gel strength and is easier to handle.

[0045] Furthermore, it can be used in combination with substances known to promote RT-PCR in the art. Useful promoting substances in the present invention include, but are not limited to, glycerol, polyols, protease inhibitors, single-strand binding proteins (SSBs), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoin, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, Triton X-100, Triton X-114, Tween 20, Nonidet P40, Briji 58, and others. Furthermore, to reduce reaction inhibition, chelating agents such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) may be included.

[0046] In the method of the present invention, it is preferable that the one-step RT-PCR reaction solution in step (3) further contains one or more primer pairs corresponding to the target region. Examples of primer pairs used in the present invention include a pair of primers corresponding to the detection region (target region) of the RNA virus to be detected, in which one primer is complementary to the DNA extension product of the other primer. Another embodiment is so-called multiplex PCR, which contains two or more pairs of the above primers. Furthermore, if the target nucleic acid consists of subtypes, degenerate primers may also be included. In the present invention, when detecting coronavirus (SARS-nCoV-2), a type of enveloped RNA virus, examples of primer pairs include the sequences (SEQ ID NOs: 1, 2, 4, 5) described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases, and the "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-pCR Panel Primers and Probes" (SEQ ID NOs: 7, 8, 10, 11, 13, 14) published by the Centers for Disease Control and Prevention (CDC), which can be suitably used in the present invention, but are not limited to these. Among the primer sequences described above, SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, and SEQ ID NOs: 13 and 14 detect the nucleocapsid protein (N) region of SARS-nCoV-2. In detecting coronaviruses, including SARS-nCOV-2, genes such as the nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, and Open Reading Frame (ORF) region can be targeted for detection, but are not limited to these. Preferably, the concentration of the forward primer is 0.1 μM to 3 μM relative to the total RT-PCR reaction solution, and the concentration of the reverse primer is 0.1 μM to 3 μM.More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less.

[0047] In another aspect, the present invention further comprises a detection method including at least one labeled hybridization probe or double-stranded DNA-binding fluorescent compound. This allows for monitoring of the amplified product by monitoring the fluorescence signal rather than by conventional electrophoresis, thereby reducing analytical effort. Furthermore, it eliminates the need to open the reaction vessel, reducing the risk of contamination. It is also possible to identify viral subtypes by labeling each hybridization probe corresponding to a viral subtype with a different fluorescent dye.

[0048] Examples of double-stranded DNA-binding fluorescent compounds include SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen® (Biotium), LCGreen (Idaho), and LightCycler® 480 ResoLight (Roche Applied Science).

[0049] Examples of hybridization probes used in the present invention include TaqMan hydrolysis probes (U.S. Patent No. 5,210,015, U.S. Patent No. 5,538,848, U.S. Patent No. 5,487,972, and U.S. Patent No. 5,804,375), molecular beacons (U.S. Patent No. 5,118,801), and FRET hybridization probes (International Publication No. 97 / 46707, International Publication No. 97 / 46712, and International Publication No. 97 / 46714). Examples of probe sequences for detecting coronavirus (SARS-nCoV-2), a type of enveloped RNA virus, include those described in "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers and Probes" (SEQ ID NOs. 9, 12, 15) published by the Centers for Disease Control and Prevention (CDC) and the sequences described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases (SEQ ID NOs. 3, 6), which can be suitably used in the present invention, but are not limited to these. The probe sequences described above detect the N region of SARS-nCoV-2. Furthermore, if the target nucleic acid consists of subtypes, degenerate sequences may also be included. In the detection of coronaviruses, including SARS-nCoV-2, genes such as the N region, E region, S region, RdRp region, and ORF region can be targeted for detection, but are not limited to these. The concentration of the fluorescently labeled probe is preferably 0.01 μM to 1.0 μM. More preferably, the concentration is 0.013 μM or more and 0.75 μM or less, and even more preferably, 0.02 μM or more and 0.5 μM or less.

[0050] Another aspect of the present invention is a kit for testing viral RNA in a sample, characterized by comprising a pretreatment solution containing a polar organic solvent and an anionic polymer, a reverse transcriptase and a DNA polymerase (or a DNA polymerase having reverse transcription activity), and a one-step RT-PCR reaction solution. The virus testing kit of the present invention comprises at least a reagent containing a polar organic solvent and an anionic polymer, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. The virus testing kit of the present invention may include a reagent containing both a polar organic solvent and an anionic polymer, or it may include the polar organic solvent and the anionic polymer as separate reagents. From the viewpoint of making the testing procedure easier, it is preferable to be provided as a reagent containing both a polar organic solvent and an anionic polymer, and a preferred kit of the present invention is provided in an embodiment that includes a reagent containing both. The one-step RT-PCR reaction solution preferably contains at least one of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol, and gelatin. Preferably, the kit includes a primer pair corresponding to the detection region of the RNA virus to be detected, and a hybridization probe corresponding to the detection region of the RNA virus to be detected. The kit of the present invention can be provided in which the above-mentioned various components are sealed in the same container or in separate containers, for example, in a single package, and in a manner that includes information on how to use the kit. By using the kit of the present invention, it is possible to quickly and easily test for the presence or absence of RNA viruses in a sample. [Examples]

[0051] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following examples.

[0052] Test Example 1. Effect of anionic polymers on RT-PCR inhibition by RNases (1) Preparation of reaction solution The reaction solution with the composition shown below was used as the basic composition, and coronavirus RNA in the reaction solution was detected in the presence of RNase A by one-step RT-PCR. Except for the pretreatment solution, the SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used as the detection reagent. The primers and probes included with this reagent are the N1 set described in the "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective: 24 Jan 2020) published by the Centers for Disease Control and Prevention (CDC). The probes used were modified with FAM as a fluorescent label and BHQ1 (Black hole quencher) as a quenching group. RT-PCR reaction solution (43 μL) Reaction solution: 30 μL Enzyme solution: 5 μL Primer / probe solution: 5 μL RNase-free water: 3 μL (2) Addition of template RNA and RNase A and pretreatment To 3 μL of 100% dimethyl sulfoxide, 1 μL of RNAse-free water or 1 ng / μL RNase A (Nacalai Tesque) was added, and 1 μL of RNAse-free water or sodium polyvinyl sulfonate (PVSA) was mixed in to a final concentration of 0.001%. Then, 2 μL of AcroMetrix Coronavirus 2019 (COVID-19) RNA Control (Thermo Fisher Scientific) was mixed in to a final concentration of 50 copies / reaction to 5 copies / reaction, and immediately 7 μL of the mixture was heat-treated in a thermal cycler at 95°C for 5 minutes. (3) Addition of reaction solution 43 μL of the RT-PCR reaction solution prepared in (1) was added to 7 μL of the heat-treated mixture from the previous step, and RT-PCR was performed in a 50 μL reaction system. (4) RT-PCR reaction conditions A real-time PCR reaction was performed using StepOne plus (Thermo Fisher Scientific) with the following temperature cycle. 42°C for 5 minutes (reverse transfer conditions) 95℃ for 10 seconds (thermal denaturation) 95°C 1 second - 50°C 3 seconds - 55°C 10 seconds, 50 cycles (PCR - fluorescence reading) (5) Results The measurement results were obtained by calculating the Ct value using the analysis software included with StepOne plus (Thermo Fisher Scientific), with a threshold of 10,000. These results are shown in Table 1 and Figure 1 below. As shown in these results, when RNase A was added, all copies from 50 to 5 were undetectable under conditions where PVSA was absent. Conversely, in the presence of PVSA, 50 to 5 copies became detectable. [Table 1]

[0053] Test Example 2. Investigation of the effects of anionic polymers using inactivated viruses. The reaction solution with the composition shown below was used as the basic composition, and inactivated coronavirus in the reaction solution was detected in the presence of RNase A by one-step RT-PCR. Except for the pretreatment solution, the SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used as the detection reagent. The primers and probes included with this reagent are the N1 set described in the "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective: 24 Jan 2020) published by the Centers for Disease Control and Prevention (CDC). The probes used were modified with FAM as a fluorescent label and BHQ1 (Black hole quencher) as a quenching group. RT-PCR reaction solution (41 μL) Reaction solution: 30 μL Enzyme solution: 5 μL Primer / probe solution: 5 μL RNase-free water: 1 μL (2) Addition of inactivated virus and RNase A and pretreatment To 3 μL of 100% dimethyl sulfoxide, 1 μL of RNAse-free water or 100 ng / μL RNase A (Nacalai Tesque) was added, and 1 μL of RNAse-free water or sodium polyvinyl sulfonate (PVSA) was mixed in to a final concentration of 0.001%. Then, 4 μL of the Positive control from the AccuPlex SARS-CoV-2 Reference Material Kit (Seracare) was added as an inactivated coronavirus sample to a final concentration of 20 copies / reaction, and 9 μL of the mixture was immediately heat-treated in a thermal cycler at 95°C for 5 minutes. (3) Addition of reaction solution 9 μL of the mixed solution after heat treatment in the preceding step was added to 41 μL of the RT-PCR reaction solution prepared in (1), and RT-PCR was performed in a 50 μL reaction system. (4) RT-PCR reaction conditions Real-time PCR reactions were performed using StepOne plus (Thermo Fisher Scientific) with the following temperature cycles. 42°C for 5 minutes (reverse transfer conditions) 95℃ for 10 seconds (thermal denaturation) 95°C 1 second - 50°C 3 seconds - 55°C 10 seconds, 50 cycles (PCR - fluorescence reading) (5) Results The measurement results were obtained by calculating the Ct value using the analysis software included with StepOne plus (Thermo Fisher Scientific), with a threshold of 10,000. These results are shown in Table 2 and Figure 2 below. As shown in these results, when RNase A was added, all 20 copies of the inactivated virus (n=4) were undetectable under conditions where PVSA was absent. Conversely, in the presence of PVSA, all copies became detectable. [Table 2]

[0054] Test Example 3. Study using saliva samples The reaction solution with the composition shown below was used as the basic composition, and inactivated coronavirus in the reaction solution in the presence of a saliva sample was detected by one-step RT-PCR. Except for the pretreatment solution, the SARS-CoV-2 Detection Kit -N1 set- (Toyobo) was used as the detection reagent. The primers and probes included with this reagent are the N1 set described in the Centers for Disease Control and Prevention (CDC) publication "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective: 24 Jan 2020). The probes used were modified with FAM as a fluorescent label and BHQ1 (Black hole quencher) as a quenching group. RT-PCR reaction solution (41 μL) Reaction solution: 30 μL Enzyme solution: 5 μL Primer / probe solution: 5 μL RNase-free water: 1 μL (2) Addition of saliva sample and pretreatment of inactivated virus 3 μL of 100% dimethyl sulfoxide was mixed with 1 μL of RNAse-free water or saliva, and then 1 μL of RNAse-free water or sodium polyvinyl sulfonate (PVSA) was added to achieve a final concentration of 0.001%. Subsequently, 4 μL of the Positive control from the AccuPlex SARS-CoV-2 Reference Material Kit (Seracare) was added as an inactivated coronavirus sample to achieve a final concentration of 20 copies / reaction, and 9 μL of the mixture was immediately heat-treated in a thermal cycler at 95°C for 5 minutes. (3) Addition of reaction solution 9 μL of the mixed solution after heat treatment in the preceding step was added to 41 μL of the RT-PCR reaction solution prepared in (1), and RT-PCR was performed in a 50 μL reaction system. (4) RT-PCR reaction conditions Real-time PCR reactions were performed using StepOne plus (Thermo Fisher Scientific) with the following temperature cycles. 42°C for 5 minutes (reverse transfer conditions) 95℃ for 10 seconds (thermal denaturation) 95°C 1 second - 50°C 3 seconds - 55°C 10 seconds, 50 cycles (PCR - fluorescence reading) (5) Results The measurement results were obtained by calculating the Ct value using the analysis software included with StepOne plus (Thermo Fisher Scientific), with a threshold of 10,000. These results are shown in Table 3 and Figure 3 below. As shown in these results, when RNase A was added, it was undetectable in all 20 copies (n=4) of the inactivated virus under conditions where PVSA was absent. Conversely, in the presence of PVSA, detection was confirmed in 3 out of 4 samples. [Table 3] [Industrial applicability]

[0055] The present invention is suitably used in molecular biological research, as well as in tests for clinical examinations and food hygiene management.

Claims

1. A method for testing for enveloped RNA viruses in a sample, characterized by comprising the following steps; (1) A step of preparing a mixture containing an RNA sample that has not been purified, an anionic polymer, and a polar organic solvent, and substantially free of surfactants. Here, the content of the anionic polymer in the mixture is 0.001% or more and 0.01% or less, and the content of the polar organic solvent is 30% or more and 75% or less. (2) A step of heating the mixture, (3) Adding to the heated mixture a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcription activity, (4) After sealing the reaction vessel, the one-step RT-PCR reaction is carried out.

2. The inspection method according to claim 1, wherein the time from preparing the mixed solution in step (1) to carrying out step (2) is 5 minutes or less.

3. The inspection method according to item 1 or 2, characterized in that the heating conditions in step (2) are 70°C or higher and for 1 second or longer.

4. The testing method according to any one of claims 1 to 3, wherein the sample is at least one selected from the group consisting of feces, pharyngeal swab, nasal swab, sputum, lung aspirate, cerebrospinal fluid, gargle solution, saliva, tears, cultured cells, culture supernatant, and environmental swab test samples.

5. The testing method according to any one of claims 1 to 4, wherein the sample is a suspension in water, physiological saline, buffer solution, or Sputazyme enzyme solution, or the supernatant or concentrate thereof obtained by centrifugation.

6. The testing method according to any one of claims 1 to 5, characterized in that the enveloped RNA virus is selected from the group consisting of Flaviviridae viruses; Togaviridae viruses; Coronavirusidae viruses; Orthomyxoviridae viruses; Rhabdoviridae viruses; Bunyaviridae viruses; Paramyxoviridae viruses; and Filoviridae viruses.

7. A testing method according to any one of claims 1 to 6, wherein the enveloped RNA virus is a coronavirus of the Coronaviridae family.

8. The testing method according to claim 7, wherein the coronavirus family virus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCoV-2 coronavirus.

9. The testing method according to any one of claims 1 to 8, characterized in that the polar organic solvent is at least one selected from the group consisting of ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pyridine, triethylamine dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, and acetonitrile.

10. The inspection method according to any one of claims 1 to 9, wherein the anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphate group, a sulfate group, and a phosphonic acid group.

11. A method for testing for viruses according to any one of claims 1 to 10, wherein the anionic polymer is at least one anionic polymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin, dermatan sulfate, polyvinyl sulfonic acid, polyvinylphosphonic acid, polystyrene sulfonic acid, polyacrylic acid, polyacrylic acid / sulfonic acid copolymer, polyacrylic acid / maleic acid copolymer, and salts thereof.

12. The testing method according to any one of claims 1 to 11, characterized in that the DNA polymerase is selected from the group consisting of Taq, Tth, and their variants.

13. The testing method according to any one of claims 1 to 12, characterized in that the reverse transcriptase is selected from the group consisting of Moloney's mouse leukemia virus (MMLV), avian myeloblastosis virus (AMV), and their variants.

14. The testing method according to any one of claims 1 to 13, characterized in that the one-step RT-PCR reaction solution in step (4) further comprises at least one selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum albumin, glycerol, glycol, and gelatin.

15. The method for testing for viruses according to claim 14, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.

16. A test kit for enveloped RNA viruses comprising a mixture containing an anionic polymer and a polar organic solvent, and a one-step RT-PCR reaction solution containing (i) reverse transcriptase and DNA polymerase or (ii) DNA polymerase having reverse transcriptase activity, wherein the content of the anionic polymer in the mixture is 0.001% or more and 0.01% or less, and the content of the polar organic solvent is 30% or more and 75% or less, and the reaction solution is substantially free of surfactants.

17. The test kit according to claim 16, further comprising at least one selected from the group consisting of betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol, and gelatin.

18. The test kit according to claim 16 or 17, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.

19. The test kit according to any one of claims 16 to 18, further comprising a hybridization probe corresponding to the detection region of the RNA virus to be detected.

20. The test kit according to any one of claims 16 to 19, characterized in that the enveloped RNA virus is selected from the group consisting of Flaviviridae viruses; Togaviridae viruses; Coronavirusidae viruses; Orthomyxoviridae viruses; Rhabdoviridae viruses; Bunyaviridae viruses; Paramyxoviridae viruses; and Filoviridae viruses.

21. The test kit according to claim 20, wherein the enveloped RNA virus is a coronavirus of the Coronaviridae family.

22. The test kit according to claim 20 or 21, characterized in that the coronavirus family virus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCoV-2.

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