JP7803271B2 - Improved virus detection methods - Google Patents

Description

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.

Coronaviruses are causative viruses of respiratory infections, including the common cold, and are said to be responsible for approximately 10 to 35% of colds during epidemic seasons. Mutant viruses are known to emerge, and in rare cases, variants that cause fatal and severe 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, it goes without saying that simple, rapid, and highly sensitive detection of coronaviruses is important in clinical diagnosis, food hygiene inspections, environmental testing, and the like.

For coronavirus pathogen testing, electron microscopy, immunological antigen detection using 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 techniques have been developed for detecting coronaviruses using nucleic acid amplification (e.g., Non-Patent Document 1, Non-Patent Document 2, Patent Document 1).

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 genomic 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, 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 cumbersome and require a lot of work time.

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, by 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 demand for the development of methods for testing coronaviruses, particularly SARS-nCOV-2, that reduce the risk of infection to medical personnel performing the tests by reducing and simplifying the work processes as much as possible.

Patent Publication No. 2012-24039

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2005, p. 5452-5456 J Virol Methods. 2004 Sep 1;120(1):33-40. Published Online January 29, 2020, https://doi. org/10.1016/S0140-6736(20)30251-8 World Health Organization (WHO) website (Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR) National Institute of Infectious Diseases website: "Pathogen Detection Manual 2019-nCoV" (https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200217.pdf) J. Animal Science and Biotechnology, Volume 5, 2014, Page 45

The present invention has been made in view of the problems of the conventional technology, and aims to enable the detection of the presence or absence of RNA of enveloped viruses, particularly coronaviruses (especially SARS-nCOV-2), simply and quickly by one-step RT-PCR without prior isolation or purification of nucleic acids from a sample.

In view of the above circumstances, the present inventors have conducted extensive research and have found 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, thereby completing the present invention.

Representative aspects of the present invention are as follows:

Item 1. A method for detecting an enveloped RNA virus in a sample, the method 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), the 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 containing no 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 centrifugal supernatant of the suspension.

Item 11. The 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 detection 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 a "betaine-like quaternary ammonium salt"), 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. The 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 polar solvent is mixed with the sample and the reagent containing the polar solvent so that the final concentration of the polar solvent in the mixed solution is adjusted to 20% or more.

A testing kit for seroconverters.

Item 23. The 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. The 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 detection kit according to Item 19 or 24, further comprising a primer pair corresponding to a detection region of an RNA virus to be detected.

Item 26. The virus detection 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. The virus testing kit according to any one of Items 19 to 26, wherein the enveloped RNA virus is a coronavirus.

Item 28. The 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.

The present invention makes it possible to easily and quickly detect 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 procedures, allowing for an increased number of tests, contributing to the early detection of infectious diseases and the prevention of their spread. Furthermore, by omitting the viral RNA purification step, the process of opening and closing the reaction vessel lid is also eliminated. 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 procedures. Furthermore, eliminating the risk of virus-containing samples scattering also reduces the risk of infection for workers.

In particular, the present invention is highly 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, 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 the like.

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, "to" means "more than or equal to, less than or equal to," and for example, if the specification states "X to 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, the singular expression should be understood to include the concept of the plural form, unless otherwise specified.

One aspect of the present invention is a method for testing for an enveloped RNA virus (herein also referred to as an "enveloped RNA virus", etc.) such as a coronavirus (particularly SARS-nCOV-2) in a sample, which includes 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 previously separating and purifying viral RNA from the sample.

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.

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.

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 viruses (e.g., SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus), Flaviviridae viruses (e.g., hepatitis C virus, Japanese encephalitis virus, Zika virus), Togaviridae viruses (e.g., rubella virus, chikungunya virus), Orthomyxoviridae viruses (e.g., influenza virus), Rhabdoviridae viruses (e.g., rabies virus), Bunyaviridae viruses (e.g., Crimean-Congo fever virus), Paramyxoviridae viruses (e.g., measles virus, human respiratory syncytial virus), and Filoviridae viruses (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.

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, rectal feces), vomit, and saliva. These samples are particularly useful for detection of HIV-1 in throat swabs, nasal swabs, sputum, saliva, pulmonary aspirates, and feces (excreted feces, 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 present 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 they 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 high levels 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.

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.

Another example of a sample in the present invention is a swab test sample. Swab tests are useful for clarifying 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, samples obtained by wiping a relevant section or facility with a cotton swab or the like, eluting the sample in water or a buffer solution, and concentrating the sample using polyethylene glycol (PEG) precipitation or the like. Specific examples of swab test procedures include "Improved Norovirus Testing Method for Swab Specimens" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html), but are not particularly limited thereto and include a wide range of methods similar thereto. Examples of areas that can be wiped include cooking 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, living rooms, etc. Also, although not a wipe test, it can be applied to concentrated sewage samples as an environmental test.

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 (especially polar organic solvents) on virus capsid structures 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.

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 more reliably exerting a high effect, it is preferable to use a polar organic solvent, and in particular, it is preferable to use methanol, triethylamine, dimethyl sulfoxide, and acetone. Furthermore, a mixed solution containing two or more of these polar organic solvents may be used, or a mixed solution of a polar organic solvent and a polar inorganic solvent may be used. When using such a polar organic solvent, the lower limit of its concentration, which is also expected to function as a denaturant for the capsid protein, is not particularly limited, although it depends on the type of polar organic solvent and other additives, as long as it is a concentration at which the capsid protein is denatured. 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 may 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.

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 destroyed 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.

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 example results described below, it has been found that up to 8% dimethyl sulfoxide can be carried over into the RT-PCR solution.

The sample transfer or heat treatment process requires the opening and closing of reaction vessels. The opening and closing of reaction vessels is cumbersome and increases 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 an infection risk for workers. Therefore, a method for testing the presence or absence of viruses using RT-PCR, which does not require the opening and closing of a lid, is significant in more than just simplifying the work process.

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.

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 thereof include Moloney Murine Leukemia Virus (MMLV)-RT, Avian Myeloblastosis Virus (AMV-RT), HIV-RT, RAV2-RT, EIAV-RT, and Carboxydothermus hydrogenoformam DNA polymerase, as well as 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.

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. More preferably, Taq, Tth, or mutants thereof are used. Particularly preferably, Tth or mutants thereof are used. 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.

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. Here, the amino acid sequence identity can be calculated by any means known in the art. For example, it can be calculated using an analytical tool that is commercially available or available via telecommunications lines (Internet), such as the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http://www.ncbi.nlm.nih. The amino acid sequence identity can be calculated by using default (initial setting) parameters in .gov/BLAST/. 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" can 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.

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.

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.

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.

Furthermore, the additive contained in the one-step RT-PCR reaction solution preferably includes 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.

Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine, but are not particularly limited as long as they are quaternary ammonium salts 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 structure. Furthermore, they are 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, more preferably 0.2 M to 1.2 M.

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. In 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.

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 concentration used is preferably a level that stabilizes PCR amplification while not interfering with fluorescence detection. It is preferably 1 to 5%, and more preferably 1 to 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.

Furthermore, the RT-PCR promoter may be used in combination with a substance known in the art to promote RT-PCR. Examples of promoters 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 acetic acid-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.

The primer pair used in the present invention is a primer pair corresponding to the detection region of the RNA virus to be detected, 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, in which two or more pairs of the above primers are included. Furthermore, if the target nucleic acid consists of 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, and these sequences can also be suitably used in the present invention, but are not limited thereto. 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 such that the forward primer concentration is 0.1 μM to 3 μM and the reverse primer concentration is 0.1 μM to 3 μM 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.

Another aspect of the present invention is 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.

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).

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 base sequence of a coronavirus detection probe include the sequences (SEQ ID NOS: 3 and 6) described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases, and these sequences are suitable for use in the present invention, but are not limited thereto. The probe sequences described above detect the N region of SARS-nCOV-2. Furthermore, when 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 or more and 1.0 μM or less, more preferably 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.

Another aspect of the present invention is a kit for testing for viral RNA in a sample, specifically for enveloped RNA viruses, comprising a pretreatment solution containing a polar solvent (preferably a pretreatment solution containing 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 testing 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 during use, the final concentration of the polar solvent in the resulting mixture is 20% or higher. 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. It is preferable that the one-step RT-PCR reaction solution 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.

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 treatment. 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 used as a test kit for SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2.

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

Test Example 1: Examination of the amount of organic solvent brought in

(1) Preparation of reaction solution

Using the reaction solution with the basic composition shown below, coronavirus RNA in the reaction solution was detected by one-step RT-PCR. All components other than 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 described 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 solution (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: 2μL

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: 3μL

(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 give a final concentration of 5,000 to 50 copies/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 a Roche Light Cycler 96 under the following temperature cycles.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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%.

Test Example 2. Detection study in the presence of negative fecal suspension

(1) Preparation of reaction solution

Using the reaction solution with the basic composition shown below, coronavirus RNA in the reaction solution was detected by one-step RT-PCR. All components other than 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 described 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 solution (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: 2μL

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: 3μL

(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 give 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. 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.

(4) RT-PCR reaction conditions

This was subjected to the following temperature cycles using a Roche Light Cycler 96:

Real-time PCR reactions were performed.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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 test results demonstrated that SARS-nCOV-2 can be detected with sufficient sensitivity by a one-step RT-PCR reaction, even when samples containing impurities that inhibit the RT-PCR reaction are used as is without prior nucleic acid separation and purification treatment.

Test Example 3. Examination of the amount of negative fecal suspension

(1) Preparation of reaction solution

Using the reaction solution with the basic composition shown below, coronavirus RNA in the reaction solution was detected by one-step RT-PCR. All components other than 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 described 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 solution (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: 2.5μL

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: 3μL

(2) Pretreatment of negative fecal suspension

1 μL to 5 μL of the 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 5,000 to 50 copies/reaction, and 42 μL of the RT-PCR reaction solution prepared in (1) was immediately added. Then, RNAse-free water was added to make the reaction system 50 μL, and RT-PCR was performed. All of the above steps 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 a Roche Light Cycler 96 under the following temperature cycles.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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 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.

Test Example 4. Detection in the presence of other biological samples

(1) Preparation of reaction solution

Using the reaction solution with the basic composition shown below, coronavirus RNA in the reaction solution was detected by one-step RT-PCR. All components other than 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 described 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 solution (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: 2μL

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: 3μL

(2) Pretreatment of each specimen

1 μL each of throat swab, nasal swab, sputum treatment, and cell culture medium was added to 3 μL of 100% dimethyl sulfoxide and mixed. Each sample was prepared as follows. Cell culture medium was D-MEM (Thermo Fisher Scientific) + 5% fetal bovine serum (FBS) (Thermo Fisher Scientific). For throat swab and nasal swab, the relevant areas were swabbed and suspended in UTM virus transport medium (Copan). For sputum treatment, 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. Note that all of the 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.

(4) RT-PCR reaction conditions

This was subjected to real-time PCR reaction using a Roche Light Cycler 96 under the following temperature cycles.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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. The results of this test demonstrated 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 without prior nucleic acid separation and purification treatment, even when samples containing large amounts of impurities that inhibit RT-PCR are used as is.

Test Example 5. Detection of inactivated viruses

(1) Preparation of reaction solution

Using the reaction solution with the composition shown below as the basic composition, inactivated SARS-CoV-2 virus in the reaction solution was detected in one-step RT-PCR. All materials used, except for the primers and probe, were included with the Norovirus Detection Kit G1/G2 - High-Speed Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those described 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 solution (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: 6μL

(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 a concentration of 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 operations 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 CFX96 DEEP WELL under the following temperature cycle.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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 SARS-nCOV-2 virus could be detected under conditions where pretreatment with dimethyl sulfoxide was performed.

Test Example 6. Detection of inactivated viruses in the presence of a sample

(1) Preparation of reaction solution

Using the reaction solution with the composition shown below as the basic composition, inactivated SARS-CoV-2 virus was detected in the reaction solution in the presence of various specimens in one-step RT-PCR. All materials used, except for the primers and probe, were included with the Norovirus Detection Kit G1/G2 - High-Speed Probe Detection Quick Step (Toyobo). The primers and probe sequences used were those described 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 solution (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: 4μL

(2) Pretreatment of inactivated virus in the presence of a sample

1 μL of inactivated SARS-nCoV-2 virus (Inactivated SARS-CoV-2 (2019-nCoV/USA-WA1/2020) (ATCC)) prepared to 25 copies/μL, 1 μL each of saliva, throat swab, nasal swab, fecal suspension supernatant, and cell culture medium were added to 2 μL of 100% dimethyl sulfoxide 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 operations 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 even once. Note that 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 recover the supernatant, which served as the specimen. Throat and nasal swabs were collected from the appropriate sites using swabs and suspended in UTM virus transport medium (Copan). Fecal suspension supernatant was prepared by suspending feces in sterile water to prepare a 10% fecal suspension, followed by centrifugation (15,000 rpm, 5 minutes), and the supernatant was recovered. D-MEM medium supplemented with 10% heat-inactivated FBS was used as the cell culture medium. The negative control for the pretreatment solution was 2 μL of RNAse-free water.

(3) RT-PCR reaction conditions

This was subjected to real-time PCR reaction using Bio-Rad CFX96 DEEP WELL under the following temperature cycle.

42°C 10 minutes (reverse transcription conditions)

95℃ 1 minute (thermal denaturation)

98°C 15 seconds - 55°C 15 seconds - 60°C 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 SARS-nCOV-2 virus can be detected by pretreatment with dimethyl sulfoxide even in the presence of various specimens.

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 (24)

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. 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. 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. 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. 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. 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. The testing method according to claim 1, wherein the sample is a suspension in advance in water, physiological saline, or a buffer solution. 8. The testing method according to claim 7 , wherein the sample is a centrifugal supernatant of the suspension. 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. 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. 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. 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. 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. 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. The method for testing according to claim 14 , wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. A kit for detecting enveloped viruses , for use in the method of claim 1, comprising a reagent containing dimethyl sulfoxide , a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. 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. A virus testing kit according to claim 16 or 17, wherein the sample and the reagent containing dimethyl sulfoxide are mixed together to prepare a mixed solution in which the final concentration of dimethyl sulfoxide in the mixed solution is adjusted to 20% or more. 19. The virus detection kit according to any one of claims 16 to 18 , wherein the reagent containing dimethyl sulfoxide does not contain a surfactant. 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. 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. 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. A virus testing kit according to any one of claims 16 to 22 , wherein the enveloped RNA virus is a coronavirus. 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.