Improved virus detection methods

Abstract

The purpose of the present invention is to provide a method and a kit by which the presence of an enveloped RNA virus (for example, a corona virus) in a sample can be easily detected within a short period of time. A method for testing an enveloped RNA virus in a sample, said method being characterized by comprising steps (1) to (3): (1) a step for mixing a sample, which has not been subjected to a nucleic acid separation and purification treatment, with a reagent containing a polar solvent; (2) a step for adding, to the liquid mixture obtained above, a one-step RT-PCR reaction solution containing (i) a reverse transcriptase and a DNA polymerase, or (ii) a DNA polymerase having reverse transcription activity; and (3) a step for sealing the reaction container and then performing a one-step RT-PCR reaction.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting viruses by nucleic acid amplification. More specifically, it relates to the detection of enveloped RNA viruses by mixing a sample with a polar solvent and then adding a reaction solution for real-time reverse transcription polymerase chain reaction (qRT-PCR) without isolating and purifying nucleic acids from the sample. Even more specifically, it relates to a method for detecting coronaviruses. The present invention enables highly sensitive detection of coronaviruses contained in biological samples such as throat swabs, nasal swabs, and sputum, as well as fecal samples, blood samples, and environmental swabs. The present invention can be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, and the like. [Background technology]

[0002] Coronaviruses are causative viruses of respiratory infections, including the common cold, and are said to be responsible for approximately 10-35% of colds during epidemic seasons. Mutant viruses are known to emerge, and in rare cases, variants that cause severe and potentially fatal respiratory diseases, such as SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and novel coronavirus disease (COVID-19) coronavirus (SARS-nCOV-2), have been known to emerge. Therefore, simple, rapid, and highly sensitive detection of coronaviruses is important for clinical diagnosis, food hygiene inspections, environmental testing, and other areas.

[0003] For coronavirus pathogen testing, electron microscopy, immunological antigen detection by ELISA, and viral gene detection methods 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 technologies have been developed for detecting coronaviruses using nucleic acid amplification (e.g., Non-Patent Document 1, Non-Patent Document 2, Patent Document 1).

[0004] For the mutated coronavirus SARS-nCOV-2, whose outbreak was confirmed in Wuhan, Hubei Province, China in 2019, a testing method using nucleic acid amplification technology was established as soon as analysis of the viral genome RNA was completed (e.g., Non-Patent Document 3, Non-Patent Document 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, detecting coronaviruses contained in a sample involves steps of extracting and purifying viral RNA from the sample. The steps of extracting and purifying viral RNA are complicated and require a lot of work time.

[0005] Previously, K. Kang et al. reported that highly pathogenic North American porcine reproductive and respiratory syndrome virus RNA can be detected directly from swine serum samples by RT-PCR (Non-Patent Document 6). However, these methods omit the RNA extraction and purification steps, resulting in RT-PCR inhibitors present in the sample being carried over into the reaction solution. RT-PCR inhibitors vary significantly depending on the type of sample. For example, fecal samples carry PCR inhibitors such as polysaccharides. Furthermore, it is known that the conditions for virus inactivation and RNA extraction vary significantly depending on the virus species. However, there is currently no known method for detecting coronaviruses, particularly SARS-nCOV-2, using RT-PCR from biological samples such as throat and nasal swabs and fecal samples, or environmental swabs, without the need for RNA isolation and purification steps, allowing for simple and rapid detection of these viruses. Furthermore, there is a strong need to develop methods for testing coronaviruses, particularly SARS-nCOV-2, that reduce the risk of infection to medical workers performing the tests by reducing and simplifying the work processes as much as possible. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2012-24039 [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 January 29,2020,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 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention was made in response to the problems of the prior art. Specifically, the present invention enables the detection of the presence or absence of RNA of enveloped viruses, particularly coronaviruses (especially SARS-nCOV-2), by one-step RT-PCR in a simple and rapid manner without prior isolation or purification of nucleic acids from a sample. [Means for solving the problem]

[0009] In view of the above circumstances, the present inventors conducted extensive research and discovered that enveloped viruses, particularly coronaviruses (especially SARS-nCOV-2), contained in a sample can be detected with sufficient sensitivity by mixing the sample with a polar solvent and then subjecting the sample to one-step RT-PCR, without prior nucleic acid isolation and purification. This led to the present invention.

[0010] Representative aspects of the present invention are as follows: Item 1. A method for detecting an enveloped RNA virus in a sample, comprising the following steps: (1) mixing a sample that has not been subjected to nucleic acid separation and purification treatment with a reagent containing a polar solvent; (2) adding to the mixture a one-step RT-PCR reaction solution containing (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; (3) After sealing the reaction vessel, a one-step RT-PCR reaction is carried out. Item 2. The testing method according to Item 1, wherein steps (1) to (3) are carried out in the same container. Item 3. The testing method according to Item 1 or 2, wherein after the reaction vessel is sealed in step (3), a one-step RT-PCR reaction is carried out without ever opening or closing the lid. Item 4. The testing method according to any one of Items 1 to 3, wherein the polar solvent is a polar organic solvent. Item 5. The testing method according to any one of Items 1 to 4, wherein in step (1), the final concentration of the polar solvent in the mixture of the sample and the reagent containing the polar solvent is 20% or more. Item 6. The testing method according to any one of Items 1 to 5, wherein in step (1), the reagent containing a polar solvent is a reagent that does not contain a surfactant. Item 7. The testing method according to any one of Items 1 to 6, wherein the time from mixing the sample with the reagent containing a polar solvent in step (1) to performing step (2) is less than 3 minutes. Item 8. The testing method according to any one of Items 1 to 7, wherein the sample is at least one selected from the group consisting of feces, throat swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, gargle, saliva, tears, cultured cells, and culture supernatant. Item 9. The testing method according to any one of Items 1 to 8, wherein the sample is a suspension pre-suspended in water, physiological saline, or a buffer solution. Item 10. The testing method according to Item 9, wherein the sample is a centrifuged supernatant of a suspension. Item 11. A testing method according to any one of Items 1 to 10, wherein the sample is a sample obtained by pre-suspending an environmental swab test sample in water, physiological saline, or a buffer solution, and concentrating the suspension. Item 12. The testing method according to any one of Items 1 to 11, wherein the enveloped RNA virus is a coronavirus. Item 13. The testing method according to Item 12, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2 coronavirus. Item 14. The testing method according to any one of Items 1 to 13, wherein the polar solvent is at least one polar organic solvent 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 15. The testing method according to any one of Items 1 to 14, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth, and mutants thereof. Item 16. The testing method according to any one of Items 1 to 15, wherein the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV)-derived reverse transcriptase, avian myeloblastosis virus (AMV)-derived reverse transcriptase, and mutants thereof. Item 17. The testing method according to any one of Items 1 to 16, wherein the one-step RT-PCR reaction solution in step (3) 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. Item 18. The testing method according to Item 17, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. Item 19. A kit for detecting enveloped viruses, comprising a reagent containing a polar solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. Item 20. A virus testing kit according to Item 19, which is used to test for the presence or absence of enveloped viruses in a sample that has not been subjected to nucleic acid separation and purification treatment. Item 21. The virus testing kit according to Item 19 or 20, wherein the polar solvent is a polar organic solvent. Item 22. The virus according to any one of Items 19 to 21, wherein the final concentration of the polar solvent in the mixture obtained by mixing the sample with the reagent containing the polar solvent is adjusted to 20% or more. A testing kit for seroconverters. Item 23. A virus testing kit according to any one of Items 19 to 22, wherein the reagent containing a polar solvent is a reagent containing no surfactant. Item 24. A virus testing kit according to any one of Items 19 to 23, wherein the one-step RT-PCR reaction solution contains at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin, glycerol, glycol, and gelatin. Item 25. The virus testing kit according to Item 19 or 24, further comprising a primer pair corresponding to a detection region of the RNA virus to be detected. Item 26. A virus testing kit according to any one of Items 19 to 25, further comprising a hybridization probe corresponding to a detection region of the RNA virus to be detected. Item 27. A virus testing kit according to any one of Items 19 to 26, wherein the enveloped RNA virus is a coronavirus. Item 28. A virus testing kit according to Item 27, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2. [Effects of the Invention]

[0011] The present invention enables the simple and rapid detection of the presence or absence of enveloped viruses, such as coronaviruses, in a sample, without requiring prior nucleic acid isolation and purification from the sample. Simply mixing the sample with a polar solvent and adding it to a one-step RT-PCR reaction solution is sufficient. This further improves the efficiency of testing, allowing for an increased number of tests, contributing to the early detection of infectious diseases and the prevention of their spread. Furthermore, omitting the viral RNA purification step also eliminates the need to open and close the reaction vessel lid. This eliminates the risk of virus-containing samples scattering when opening and closing the lid, reducing the risk of contamination of other samples. This also reduces the risk of false positives and further improves the accuracy of testing. Furthermore, eliminating the risk of virus-containing samples scattering also reduces the risk of infection for workers.

[0012] The present invention is particularly effective in testing samples that may contain SARS-nCOV-2 (also referred to as SARS-CoV-2, 2019-nCoV, etc.), which emerged in 2019. For example, the present invention enables highly sensitive detection of coronaviruses (particularly SARS-nCOV-2) from biological samples containing many impurities, including blood, feces (excreted feces and rectal feces), vomit, urine, sputum, lymph, plasma, ejaculate, pulmonary aspirate, cerebrospinal fluid, throat swab, nasal swab, gargle, saliva, and tears, as well as environmental swab samples and samples containing cultured cells or culture supernatants. The present invention can also be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, and other applications. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in further detail below with reference to embodiments thereof, but the present invention is not limited thereto. It should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Furthermore, all non-patent documents and patent documents described in this specification are incorporated herein by reference. In this specification, "~" means "more than or equal to, less than or equal to," and for example, if the specification states "X~Y," it means "more than or equal to X, less than or equal to Y." Furthermore, in this specification, "and / or" means either one or both. Furthermore, in this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified.

[0014] One aspect of the present invention is a method for testing for enveloped RNA viruses (herein also referred to as "enveloped RNA viruses") such as coronaviruses (particularly SARS-nCOV-2) in a sample, which involves mixing the sample with a reagent containing a polar solvent, and adding a one-step RT-PCR reagent containing (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase with reverse transcription activity, without prior isolation and purification of viral RNA from the sample.

[0015] The method of the present invention for detecting the presence or absence of an enveloped RNA virus in a sample comprises: The inspection method is characterized by including at least the following steps: (1) mixing a sample that has not been subjected to nucleic acid separation and purification treatment with a reagent containing a polar solvent; (2) adding to the mixture a one-step RT-PCR reaction solution containing (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; (3) After sealing the reaction vessel, a one-step RT-PCR reaction is carried out. It is preferable that steps (1) and (2), preferably all of steps (1) to (3), are performed in the same container. That is, it is preferable that all or part of the mixed solution is not transferred to another container between steps (1) and (2), preferably between each of steps (1) to (3). The entire amount of the mixed solution obtained in step (1) may be subjected to step (2), or part of the mixed solution may be transferred to another container and then step (2) is performed. Furthermore, in step (3), it is preferable that the lid of the reaction vessel is not opened or closed after the reaction vessel is sealed.

[0016] In one embodiment, the mixture obtained in step (1) (preferably a mixture of a sample that has not been subjected to nucleic acid separation and purification treatment and a reagent containing a polar organic solvent) may be allowed to stand for approximately 1 second to 20 minutes before being subjected to step (2), or may not be allowed to stand for approximately 1 second to 20 minutes. From the viewpoint of performing an enveloped RNA virus test in a shorter time, the time from obtaining the mixture in step (1) to performing step (2) is preferably less than 10 minutes, more preferably less than 5 minutes, even more preferably less than 3 minutes, even more preferably less than 2 minutes, and particularly preferably less than 1 minute. In this case, the lower limit is not particularly limited as long as the effects of the present invention are achieved. After obtaining the mixture in step (1), step (2) may be performed immediately, or step (2) may be performed after, for example, 1 second or more, preferably 5 seconds or more, more preferably 10 seconds or more have elapsed. According to the present invention, it has been confirmed that enveloped RNA viruses in a sample can be detected with sufficient sensitivity even when the time for mixing and reacting the sample with a reagent containing a polar solvent (preferably a polar organic solvent) is short.

[0017] The RNA virus to be tested in the present invention is not particularly limited as long as it has an envelope derived from a lipid bilayer. Examples of such enveloped RNA viruses include Coronaviridae (e.g., SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus), Flaviviridae (e.g., Hepatitis C virus, Japanese encephalitis virus, Zika virus), Togaviridae (e.g., Rubella virus, Chikungunya virus), Orthomyxoviridae (e.g., Influenza virus), Rhabdoviridae (e.g., Rabies virus), Bunyaviridae (e.g., Crimean-Congo fever virus), Paramyxoviridae (e.g., Measles virus, Human Respiratory Syndrome virus), and Filoviridae (e.g., Ebola virus). These viruses are particularly useful for detecting coronaviruses, particularly SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2 coronavirus.

[0018] Examples of samples that can be used in the present invention include, but are not limited to, throat swabs, nasal swabs, sputum, feces (excreted feces and rectal feces), vomit, and saliva. These samples are particularly useful for detecting HIV-1 in throat swabs, nasal swabs, sputum, saliva, pulmonary aspirates, and feces (excreted feces and rectal feces). These samples contain contaminants such as proteases and nucleases, and feces is characterized by the presence of large amounts of Escherichia coli-derived proteins and nucleic acids. 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 contaminants in the sample, resulting in reduced detection sensitivity. One feature of the present invention is that pretreatment of these samples, such as isolating or extracting RNA using a commercially available RNA purification kit, is not required. In addition, in general, virus testing methods sometimes involve prior heat treatment or the like to expose viral nucleic acids before performing a nucleic acid amplification reaction. From the perspective of more easily and quickly testing enveloped RNA viruses, a preferred embodiment of the present invention does not require prior heat treatment of such samples. These samples may be subjected to detection directly, or may be suspended in water, physiological saline, or a buffer solution to reduce the influence of contaminants on the reaction and obtain more stable test results. Furthermore, for samples containing particularly large amounts of contaminants, such as feces, centrifugal separation may be performed and the resulting supernatant may be used. Alternatively, filtration may be performed. Examples of buffer solutions include, but are not limited to, Hank's buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and Tricine buffer.

[0019] Another example of a sample according to the present invention is a sample containing cultured cells or a culture supernatant. Cell-based isolation and culture is effective for isolating viruses. The culture supernatant and cultured cells after isolation and culture contain viruses, and thus can serve as samples according to the present invention. Cell types that can be used for isolation and culture include MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells, and Sf21 cells, but are not particularly limited thereto, and methods similar thereto are broadly encompassed.

[0020] Another example of a sample in the present invention is a swab test sample. Swab tests are useful for elucidating contamination routes and understanding the contamination status of facility environments, etc. In the present invention, swab tests are not particularly limited, but include, for example, a sample obtained by wiping a relevant area or equipment with a cotton swab or the like, eluting it in water or a buffer solution, and concentrating it using polyethylene glycol (PEG) precipitation. Specific swab test procedures include "Improved Norovirus Testing Method for Swab Samples" (http: / / idsc.nih.go.jp / iasr / 32 / 382 / dj3824.html), but are not particularly limited and include a wide range of methods similar to this. Examples of areas to be swabbed include kitchen utensils such as cutting boards, knives, dishcloths, and tableware; refrigerator handles, toilet and bathroom doorknobs; faucets in the washroom, kitchen, toilet, and bathroom; the hands and fingers of cooks; bathrooms, toilets, washbasins, handrails; and living rooms. Although it is not a swab test, it can also be applied to concentrated sewage samples as an environmental test.

[0021] In the present invention, polarity refers to the electronic imbalance present within a molecule, and a molecule in which the center of gravity of the positive and negative charges within the molecule does not coincide is called a polar molecule. A solvent composed of polar molecules is called a polar solvent. Among polar solvents, polar organic solvents composed of organic compounds are preferred because they can destabilize the higher-order structure of biomolecules such as nucleic acids and proteins, weaken the hydrophobic bonds of viral structural proteins, and are expected to have the effect of destabilizing the capsid structure. As mentioned above, it is known that the destabilizing effect of polar solvents (particularly polar organic solvents) on the capsid structure of viruses varies depending on the type of virus. This is thought to be due to differences in the strength of hydrophobic bonds and other properties due to differences in the properties of the capsid proteins possessed by viruses.

[0022] Specific examples of the polar solvent include polar organic solvents such as 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, and pyridine; and polar inorganic solvents such as water, but are not limited thereto. From the viewpoint of ensuring a high level of effectiveness, polar organic solvents are preferably used, and methanol, triethylamine, dimethyl sulfoxide, and acetone are particularly preferred. A mixed solution containing two or more of these polar organic solvents may also be used, or a mixed solution of a polar organic solvent and a polar inorganic solvent may also be used. When using a polar organic solvent that is also expected to function as a capsid protein denaturant, the minimum concentration is not particularly limited as long as it is a concentration at which the capsid protein is denatured, although this depends on the type of polar organic solvent and other additives. Furthermore, since capsid proteins differ depending on the type of virus, the effective concentration of the polar solvent (preferably a polar organic solvent) differs for each virus, but typically the final concentration of the polar solvent in the mixture of the sample and the reagent containing the polar solvent (effective concentration of the polar solvent relative to the volume of the specimen) is 10% or more, preferably 15% or more, more preferably 20% or more, even more preferably 30% or more, and particularly preferably 50% or more. In this case, the upper limit of the final concentration of the polar solvent is not particularly limited as long as it does not inhibit the effects of the present invention, but can be, for example, less than 100%, preferably 95% or less, more preferably 90% or less, even more preferably 85% or less, and particularly preferably 80% or less.

[0023] The polar solvent may be used in combination with one or more surfactants, reducing agents, chelating agents, and metal salts, or may not be used in combination with these. In virus testing methods, the viral envelope may be disrupted in advance with a pretreatment solution containing a surfactant before a nucleic acid amplification reaction is performed. In the present invention, enveloped RNA viruses in a sample can be detected with sufficient sensitivity without pretreatment using a reagent containing such a surfactant.

[0024] Among the polar solvents, polar organic solvents are generally known to be inhibitors of PCR. Therefore, by selecting a polar organic solvent with a small difference between the concentration required for protein denaturation and the allowable concentration for carryover into PCR, the polar organic solvent, sample, and one-step RT-PCR reaction solution can be added sequentially, allowing for a simple detection procedure from capsid protein denaturation to the one-step RT-PCR reaction in the same container without opening and closing the container. A particularly preferred example of such a polar organic solvent is dimethyl sulfoxide. For example, when 1 μL of dimethyl sulfoxide and 1 μL of sample are mixed and 48 μL of one-step RT-PCR solution is added, the concentration of dimethyl sulfoxide carried over into the reaction solution is 2%. 2% dimethyl sulfoxide is an allowable concentration for carryover into the RT-PCR solution. Furthermore, as shown in the test results described below, it has been found that up to 8% dimethyl sulfoxide can be carried over into the RT-PCR solution.

[0025] Sample transfer or heat treatment processes require the opening and closing of reaction vessels. This process is cumbersome and lengthens the work time. In addition, opening and closing reaction vessels containing virus-containing samples poses a risk of scattering viruses and virus-derived RNA. Virus scattering threatens the safety and health of workers and also contaminates the testing environment. Since scattered RNA viruses become aerosolized in the workplace, there is a risk of contamination of other samples being tested simultaneously and of infection to workers. For this reason, a method for testing the presence or absence of viruses using RT-PCR, which does not require the opening and closing of the lid, is significant in more than just simplifying the process.

[0026] The one-step RT-PCR solution added to the mixture contains (i) a reverse transcriptase and a DNA polymerase, or (ii) a DNA polymerase that also has reverse transcriptase activity. (ii) As the DNA polymerase with reverse transcriptase activity, Tth DNA polymerase, Taq DNA polymerase, or the like is preferably used. In one preferred embodiment, it is suitable to use two types of enzymes, for example, at least two types of enzymes, a reverse transcriptase and a DNA polymerase.

[0027] The origin of the reverse transcriptase (RT) contained in the one-step RT-PCR reaction solution is not particularly limited as long as it can convert RNA to DNA, and examples include Moloney Murine Leukemia Virus (MMLV)-RT, Avian Myeloblastosis Virus (AMV-RT), HIV-RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase, and mutants thereof. Particularly preferred examples include Moloney Murine Leukemia Virus (MMRV)-derived reverse transcriptase (MMLV-RT), Avian Myeloblastosis Virus (AMV)-derived reverse transcriptase (AMV-RT), and mutants thereof.

[0028] Examples of DNA polymerases contained 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 mutants thereof. Taq, Tth, or mutants thereof are more preferred. Tth or mutants thereof are particularly preferred. Furthermore, to enhance the effect of suppressing nonspecific reactions, it is preferable to use the DNA polymerase in combination with an anti-DNA polymerase antibody or to introduce a thermolabile blocking 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.

[0029] As used herein, a reverse transcriptase or DNA polymerase mutant refers to a mutant that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 99% or more sequence identity to the amino acid sequence of the wild-type reverse transcriptase or wild-type DNA polymerase from which it is derived, and that has the activity of amplifying DNA and / or converting RNA to cDNA, similar to the wild-type reverse transcriptase or wild-type DNA polymerase. The amino acid sequence identity can be calculated by any method known in the art. For example, it can be calculated using an analytical tool that is commercially available or available via telecommunications lines (Internet). For example, amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / with default (initial setting) parameters. Furthermore, a mutant that can be used in the present invention is a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added (hereinafter, these are collectively referred to as "mutations") in the amino acid sequence of the wild-type reverse transcriptase or wild-type DNA polymerase from which it is derived, and may have the activity of converting RNA to cDNA and / or the activity of amplifying DNA, similar to the wild-type reverse transcriptase or wild-type DNA polymerase. Here, "one or several" 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 is not particularly limited thereto.

[0030] The one-step RT-PCR reaction solution used in the present invention contains, in addition to a reverse transcriptase and a DNA polymerase, a buffer, an appropriate salt such as a magnesium salt or a manganese salt, deoxynucleotide triphosphates, and a primer pair corresponding to the target region of the viral RNA to be detected, and may further contain additives as necessary.

[0031] The buffer used in the present invention is not particularly limited, but examples thereof include Tris, Tricine, Bis-Tricine, and Bicine. The pH is adjusted to 6 to 9, more preferably 7 to 8, using sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like. The concentration of the added buffer is 10 to 200 mM, more preferably 20 to 150 mM. In this case, a salt solution is added to achieve ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and ammonium acetate.

[0032] The dNTPs used in the present invention include dATP, dCTP, dGTP, and dTTP, each added at 0.1 to 0.5 mM, most commonly about 0.2 mM. Preventive measures against cross-contamination may be taken by using dUTP instead of and / or as part of 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, and these are preferably added at about 1 to 10 mM.

[0033] Furthermore, the additives contained in the one-step RT-PCR reaction solution preferably include 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.

[0034] Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine, but there is no particular limitation as long as it is a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid. The structure of betaine-like quaternary ammonium salts is a compound with stable positive and negative charges within the molecule, which is thought to exhibit surfactant-like properties and cause destabilization of viral structures. Furthermore, they are known to promote nucleic acid amplification by DNA polymerase. The preferred concentration of the betaine-like quaternary ammonium salt is 0.1M to 2M, more preferably 0.2M to 1.2M.

[0035] The amount of bovine serum albumin added to the one-step RT-PCR reaction solution is not limited as long as the effects of the present invention are achieved, but is preferably at least 0.5 mg / ml or more, more preferably at least 1 mg / ml or more. For samples containing many impurities, a bovine serum albumin concentration of preferably 2 mg / ml or more, more preferably 3 mg / ml or more, enables good detection.

[0036] The gelatin contained in the one-step RT-PCR reaction solution is derived from the skin, bones, and tendons of animals such as cows and pigs, or the scales and skin of fish, and is thought to contribute to the stabilization of PCR enzymes. The preferred concentration is one that stabilizes PCR amplification while not interfering with fluorescence detection. It is preferably 1-5%, more preferably 1-2%. While there are no particular limitations on the origin of gelatin, fish-derived gelatin is preferred over bovine or porcine-derived gelatin because it has a lower jelly strength and allows for easier handling of the reaction solution.

[0037] Furthermore, they can be used in combination with substances known in the art to enhance RT-PCR. Examples of enhancers useful in the present invention include, but are not limited to, glycerol, polyols, protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetate-containing compounds, glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, 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, and Briji 58. Furthermore, to reduce reaction inhibition, a chelating agent such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) may be contained.

[0038] The primer pair used in the present invention is a primer pair corresponding to the detection region of the target RNA virus, and includes two pairs of primers, one of which is complementary to the DNA extension product of the other primer. Another embodiment includes so-called multiplex PCR, which includes two or more pairs of the above primers. Furthermore, if the target nucleic acid is a subtype, a degenerate primer may be included. When detecting coronavirus (SARS-nCOV-2), a type of enveloped RNA virus, in the present invention, examples of primer pairs include the sequences (SEQ ID NOS: 1 to 6) described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases, which can also be used in the present invention, but are not limited to these. Among the primer sequences described above, SEQ ID NOS: 1 and 2 and SEQ ID NOS: 4 and 5 are used to detect the nucleocapsid protein (N) region of SARS-nCOV-2. In detecting coronaviruses such as 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 detected, but are not limited to these. The concentrations of the primers used are preferably 0.1 μM to 3 μM for the forward primer and 0.1 μM to 3 μM for the reverse primer relative to the total RT-PCR reaction solution. More preferably, the forward primer concentration is 0.1 μM to 2 μM and the reverse primer concentration is 0.5 μM to 2 μM.

[0039] In another aspect, the present invention provides a detection method further comprising at least one labeled hybridization probe or double-stranded DNA-binding fluorescent compound. This allows the analysis of amplification products to be monitored by monitoring fluorescent signals rather than by conventional electrophoresis, reducing analytical labor. Furthermore, there is no need to open the reaction vessel, reducing the risk of contamination. It is also possible to identify virus subtypes by labeling each hybridization probe corresponding to a virus subtype with a different fluorescent dye.

[0040] Examples of double-stranded DNA-binding fluorescent compounds include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (registered trademark; Biotium), LCGreen (Idaho), and LightCycler (registered trademark) 480 ResoLight (Roche Applied Science).

[0041] Examples of hybridization probes used in the present invention include TaqMan hydrolysis probes (U.S. Pat. Nos. 5,210,015, 5,538,848, 5,487,972, and 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801), and FRET hybridization probes (WO 97 / 46707, WO 97 / 46712, and WO 97 / 46714). Examples of the nucleotide sequences of coronavirus detection probes include those described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases (SEQ ID NOS: 3 and 6), which can be suitably used in the present invention. The probe sequences described above detect the N region of SARS-nCOV-2. Furthermore, if the target nucleic acid is a subtype, it may contain a degenerate sequence. In detecting coronaviruses such as SARS-nCOV-2, genes in the N region, E region, S region, RdRp region, and ORF region can be detected, but are not limited to these. The concentration of the fluorescently labeled probe is preferably 0.01 μM to 1.0 μM, more preferably 0.013 μM to 0.75 μM, and even more preferably 0.02 μM to 0.5 μM.

[0042] Another aspect of the present invention is a kit for detecting viral RNA in a sample, specifically for detecting enveloped RNA viruses, comprising a pretreatment solution containing a polar solvent (preferably a polar organic solvent), a reverse transcriptase and a DNA polymerase (or a DNA polymerase with reverse transcription activity), and a one-step RT-PCR reaction solution. The virus detection kit of the present invention comprises at least a reagent containing a polar solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. The polar solvent is preferably formulated so that when the sample and the reagent containing the polar solvent are mixed at the time of use, the final concentration of the polar solvent in the mixture is 20% or more. The reagent containing the polar solvent may also be a surfactant-free reagent. The one-step RT-PCR reaction solution preferably contains at least one of a betaine-like quaternary ammonium salt, bovine serum albumin, glycerol, glycol, and gelatin. The kit preferably contains a primer pair corresponding to the detection region of the target RNA virus, and a hybridization probe corresponding to the detection region of the target RNA virus.

[0043] The virus test kit of the present invention can be used to test for the presence or absence of an enveloped virus in a sample that has not been subjected to nucleic acid isolation and purification. Such a virus test kit of the present invention can be used to test for any enveloped virus, but is preferably used as a test kit for coronaviruses, and more preferably as a test kit for SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2. [Example]

[0044] The present invention will be specifically described below with reference to examples, although the present invention is not limited to the following examples.

[0045] Test Example 1: Examination of the amount of organic solvent carried in (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, coronavirus RNA in the reaction mixture was detected by one-step RT-PCR. All components except the primers and probe were included in the Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (45 μL) Condition 1.N area 1 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL 10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL 10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL RNAse-free water: 2 μL Condition 2.N area 2 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL 10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 3 μL (2) Addition of template RNA 1 μL of coronavirus N1 RNA or N2 RNA (Nihon Gene Research Institute) was mixed with 1 to 4 μL of 100% dimethyl sulfoxide to a final concentration of 5,000 to 50 copies per reaction, and 45 μL of the RT-PCR reaction solution prepared in (1) was immediately added. RNAse-free water was added to make the reaction system 50 μL, and RT-PCR was performed. (3) RT-PCR reaction conditions This was subjected to real-time PCR reaction using Roche Light Cycler 96 with the following temperature cycles. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (4) Results The measurement results were calculated using Roche Light Cycler 96 analysis software to calculate Ct values. As a result, detection of up to 50 copies / reaction of N region 1 and N region 2 was confirmed under all conditions. It was confirmed that dimethyl sulfoxide can be introduced up to a final concentration of 8%. [Table 1]

[0046] Test Example 2: Detection study in the presence of negative fecal suspension (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, coronavirus RNA in the reaction mixture was detected by one-step RT-PCR. All components except the primers and probe were included in the Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (45 μL) Condition 1.N area 1 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL 10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL 10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL RNAse-free water: 2 μL Condition 2.N area 2 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL 10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 3 μL (2) Pretreatment of negative fecal suspension 1 μL of each of the 24 types of negative fecal suspensions was added to 3 μL of 100% dimethyl sulfoxide and mixed. (3) Addition of pretreated negative stool suspension and template RNA 4 μL of the pretreated negative fecal suspension was mixed with 1 μL of coronavirus N1 RNA or N2 RNA (Nihon Gene Research Institute) to a final concentration of 50 copies / reaction, and 45 μL of the RT-PCR reaction solution prepared in (1) was immediately added to perform RT-PCR in a 50 μL reaction system. All of the steps up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and the one-step RT-PCR reaction was carried out without opening or closing the lid. (4) RT-PCR reaction conditions This was then subjected to the following temperature cycles using a Roche Light Cycler 96: Real-time PCR reactions were performed. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (5) Results The Ct values ​​of the measurement results were calculated using Roche's Light Cycler 96 analysis software. As a result, detection of 50 copies / reaction of N region 1 and N region 2 was confirmed under all conditions. The results of this test demonstrated that SARS-nCOV-2 can be detected with sufficient sensitivity by one-step RT-PCR reaction, even when samples containing impurities that inhibit RT-PCR reaction are used as is, without prior nucleic acid isolation and purification. [Table 2]

[0047] Test Example 3: Examination of the amount of negative fecal suspension (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, coronavirus RNA in the reaction mixture was detected by one-step RT-PCR. All components except the primers and probe were included in the Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (42 μL) Condition 1.N area 1 Reaction solution: 30 μL Enzyme solution: 5 μL 20 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 1.5 μL 20 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 2 μL 10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL RNAse-free water: 2.5 μL Condition 2.N area 2 Reaction solution: 30 μL Enzyme solution: 5 μL 20 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 1.25 μL 20 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 1.75 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 3 μL (2) Pretreatment of negative fecal suspension 1 μL to 5 μL of negative fecal suspension was added to 3 μL of 100% dimethyl sulfoxide and mixed. (3) Addition of pretreated negative stool suspension and template RNA 1 μL of coronavirus N1 RNA or N2 RNA (Nihon Gene Research Institute) was mixed with the pretreated negative fecal suspension to a final concentration of 5000–50 copies / reaction, and 42 μL of the RT-PCR reaction mixture prepared in (1) was immediately added. RNAse-free water was then added to make a 50 μL reaction mixture, and RT-PCR was performed. All of the steps up to this point were carried out in the same container, and after adding the RT-PCR reaction mixture, the container was sealed and the one-step RT-PCR reaction was carried out without opening or closing the lid. (4) RT-PCR reaction conditions This was subjected to real-time PCR reaction using Roche Light Cycler 96 with the following temperature cycles. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (5) Results The measurement results were calculated using Roche Light Cycler 96 analysis software to calculate Ct values. As a result, detection of up to 50 copies / reaction of N region 1 and N region 2 was confirmed under all conditions. The test results demonstrated that SARS-nCOV-2 can be detected with sufficient sensitivity by one-step RT-PCR, even when samples containing large amounts of impurities that inhibit RT-PCR are used as is, without prior nucleic acid isolation and purification. [Table 3]

[0048] Test Example 4: Detection in the presence of other biological samples (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, coronavirus RNA in the reaction mixture was detected by one-step RT-PCR. All components except the primers and probe were included in the Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (45 μL) Condition 1.N area 1 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM N_Sarbeco_F1 (primer, SEQ ID NO: 1): 3 μL 10 μM N_Sarbeco_R1 (primer, SEQ ID NO: 2): 4 μL 10 μM N_Sarbeco_P1 (probe, SEQ ID NO: 3): 1 μL RNAse-free water: 2 μL Condition 2.N area 2 Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL 10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 3 μL (2) Pretreatment of each specimen 1 μL of throat swab, nasal swab, sputum treatment solution, and cell culture medium were each added to 3 μL of 100% dimethyl sulfoxide and mixed. Each sample was prepared as follows: D-MEM (Thermo Fisher Scientific) + 5% fetal bovine serum (FBS) (Thermo Fisher Scientific) was used as cell culture medium. For throat swab and nasal swab, the relevant areas were swabbed and suspended in UTM virus transport medium (Copan). For sputum treatment solution, an equal volume of 10% dithiothreitol was mixed and left to stand at room temperature for 15 minutes. (3) Addition of pretreated samples and template RNA 4 μL of each pretreated sample was mixed with 1 μL of coronavirus N1 RNA or N2 RNA (Nihon Gene Research Institute) to a final concentration of 50 copies / reaction, and 45 μL of the RT-PCR reaction solution prepared in (1) was immediately added to perform RT-PCR in a 50 μL reaction system. All of the steps up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and the one-step RT-PCR reaction was carried out without opening or closing the lid. (4) RT-PCR reaction conditions This was subjected to real-time PCR reaction using Roche Light Cycler 96 with the following temperature cycles. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (5) Results The measurement results were calculated using Roche Light Cycler 96 analysis software to calculate Ct values. As a result, detection of 50 copies / reaction of N region 1 and N region 2 was confirmed under all conditions. These test results demonstrate that even when using samples derived from various living organisms, SARS-nCOV-2 can be detected with sufficient sensitivity by a one-step RT-PCR reaction, even when samples containing large amounts of impurities that inhibit RT-PCR reactions are used as is, without prior nucleic acid separation and purification. [Table 4]

[0049] Test Example 5: Detection of inactivated viruses (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, inactivated SARS-CoV-2 virus in the reaction mixture was detected in a one-step RT-PCR. All components except the primers and probe were used (Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step - (Toyobo) accessories). The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (48 μL) Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL 10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 6 μL (2) Pretreatment of inactivated viruses 1 μL of inactivated SARS-nCOV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) (ATCC)) prepared to 10 copies / μL was mixed with 1 μL of 100% dimethyl sulfoxide, and 48 μL of the RT-PCR reaction solution prepared in (1) was immediately added to perform RT-PCR in a 50 μL reaction system. Note that all of the steps up to this point were carried out in the same container, and after adding the RT-PCR reaction solution, the container was sealed and the one-step RT-PCR reaction was carried out without opening or closing the lid even once. (3) RT-PCR reaction conditions This was subjected to real-time PCR reaction using Bio-Rad's CFX96 DEEP WELL under the following temperature cycles. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (4) Results The Ct values ​​of the measurement results were calculated using Bio-Rad's CFX96 analysis software. As a result, it was confirmed that the SARS-nCOV-2 virus could be detected under conditions where dimethyl sulfoxide was pretreated. [Table 5]

[0050] Test Example 6: Detection of inactivated viruses in the presence of specimens (1) Preparation of reaction solution Using the basic reaction mixture composition shown below, inactivated SARS-CoV-2 virus in the reaction mixture in the presence of various samples was detected in a one-step RT-PCR. All other components, except the primers and probe, were the Norovirus Detection Kit G1 / G2 - High-Speed ​​Probe Detection Quick Step (Toyobo) accessories. The primers and probe sequences used were those listed in the "Pathogen Detection Manual 2019-nCoV," and the probe was modified with FAM as the fluorescent label and BHQ1 (black hole quencher) as the quencher. RT-PCR reaction mixture (46 μL) Reaction solution: 30 μL Enzyme solution: 5 μL 10 μM NIID_2019-nCOV_N_F2 (primer, SEQ ID NO: 4): 2.5 μL 10 μM NIID_2019-nCOV_N_R2 (primer, SEQ ID NO: 5): 3.5 μL 10 μM NIID_2019-nCOV_N_P2 (probe, SEQ ID NO: 6): 1 μL RNAse-free water: 4 μL (2) Pretreatment of inactivated virus in the presence of the specimen 1 μL of inactivated SARS-nCoV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) (ATCC)) prepared at 25 copies / μL was added to 2 μL of 100% dimethyl sulfoxide, and 1 μL each of saliva, throat swab, nasal swab, fecal suspension supernatant, and cell culture medium were added and mixed. 46 μL of the RT-PCR reaction solution prepared in (1) was immediately added, and RT-PCR was performed in a 50 μL reaction system. Note that all steps up to this point were performed in the same container, and after adding the RT-PCR reaction solution, the container was sealed and the one-step RT-PCR reaction was performed without opening or closing the lid. Each sample was prepared as follows. Saliva was collected directly into a plastic container, and 100 μL of saliva was mixed with 100 μL of PBS in equal volumes. The mixture was then centrifuged (15,000 rpm, 5 minutes) to collect the supernatant, which was used as the specimen. Throat and nasal swabs were collected from the appropriate sites using swabs and suspended in UTM virus transport medium (Copan). For fecal suspension supernatant, feces were suspended in sterile water to prepare a 10% fecal suspension, and the supernatant was collected after centrifugation (15,000 rpm, 5 minutes). D-MEM medium supplemented with 10% heat-inactivated FBS was used as the cell culture medium. The negative control for the pretreatment liquid was 2 μL of RNAse-free water. (3) RT-PCR reaction conditions This was subjected to real-time PCR reaction using Bio-Rad's CFX96 DEEP WELL under the following temperature cycles. 42℃ 10 minutes (reverse transcription conditions) 95℃ 1 minute (thermal denaturation) 98℃ 15 seconds - 55℃ 15 seconds - 60℃ 45 seconds 50 cycles (PCR - fluorescence reading) (4) Results The measurement results were calculated using Bio-Rad's CFX96 analysis software to calculate Ct values. As a result, it was confirmed that SARS-nCOV-2 virus can be detected even in the presence of various samples by pre-treating with dimethyl sulfoxide. [Table 6] [Industrial Applicability]

[0051] The present invention is suitable for use in molecular biology research, as well as in tests for the purposes of clinical testing and food hygiene control.

Claims

1. A method for detecting an enveloped RNA virus in a sample, comprising the steps of: (1) mixing a sample that has not been subjected to nucleic acid separation and purification treatment with a reagent containing dimethyl sulfoxide; (2) adding to the mixture a one-step RT-PCR reaction solution containing (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; (3) sealing the reaction vessel and then carrying out a one-step RT-PCR reaction; An inspection method characterized in that the steps (1) to (3) are performed in the same container.

2. 2. The method according to claim 1, wherein in step (3), after the reaction vessel is sealed, the one-step RT-PCR reaction is carried out without ever opening or closing the lid.

3. 3. The testing method according to claim 1, wherein in step (1), the final concentration of dimethyl sulfoxide in the mixture of the sample and the reagent containing dimethyl sulfoxide is 20% or more.

4. 4. The testing method according to claim 1, wherein in step (1), the reagent containing dimethyl sulfoxide is a reagent that does not contain a surfactant.

5. 5. The testing method according to claim 1, wherein the time from mixing the sample with the reagent containing dimethyl sulfoxide in step (1) to performing step (2) is less than 3 minutes.

6. 6. The testing method according to claim 1, wherein the sample is at least one selected from the group consisting of feces, pharyngeal swabs, nasal swabs, sputum, pulmonary aspirates, cerebrospinal fluid, gargle, saliva, tears, cultured cells, and culture supernatant.

7. 7. The testing method according to claim 1, wherein the sample is a suspension in advance in water, physiological saline, or a buffer solution.

8. 8. The testing method according to claim 7, wherein the sample is a centrifugal supernatant of the suspension.

9. 9. The testing method according to claim 1, wherein the sample is a concentrated suspension of an environmental swab test sample pre-suspended in water, physiological saline or a buffer solution.

10. The method for detecting an enveloped RNA virus according to any one of claims 1 to 9, wherein the enveloped RNA virus is a coronavirus.

11. The testing method according to claim 10, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2 coronavirus.

12. 12. The method according to claim 1, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth, and mutants thereof.

13. 13. The method for testing according to any one of claims 1 to 12, wherein the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV)-derived reverse transcriptase, avian myeloblastosis virus (AMV)-derived reverse transcriptase, and mutants thereof.

14. 14. The testing method according to any one of claims 1 to 13, wherein the one-step RT-PCR reaction solution in step (3) 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.

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

16. A kit for testing for enveloped viruses, for use in the method of claim 1, comprising a reagent containing dimethyl sulfoxide, reverse transcriptase, DNA polymerase, and a one-step RT-PCR reaction solution.

17. 17. The virus testing kit according to claim 16, which is used to test for the presence or absence of an enveloped virus in a sample that has not been subjected to nucleic acid separation and purification treatment.

18. 18. A virus testing kit according to claim 16 or 17, wherein the final concentration of dimethyl sulfoxide in the mixed solution obtained by mixing the sample with the reagent containing dimethyl sulfoxide is adjusted to be 20% or more.

19. A virus testing kit according to any one of claims 16 to 18, wherein the reagent containing dimethyl sulfoxide is a reagent that does not contain a surfactant.

20. The virus testing kit according to any one of claims 16 to 19, wherein the one-step RT-PCR reaction solution contains at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin, glycerol, glycol, and gelatin.

21. 21. The virus testing kit according to claim 16 or 20, further comprising a primer pair corresponding to a detection region of the RNA virus to be detected.

22. 22. A virus testing kit according to any one of claims 16 to 21, further comprising a hybridization probe corresponding to a detection region of the RNA virus to be detected.

23. 23. A virus testing kit according to any one of claims 16 to 22, wherein the enveloped RNA virus is a coronavirus.

24. The virus testing kit according to claim 23, wherein the coronavirus is SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, or SARS-nCOV-2.

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