JPWO2018198682A1 - Virus testing method and virus testing kit - Google Patents

Abstract

Provided are a method and a kit that enable detection of the presence or absence of non-enveloped virus RNA by one-step RT-PCR in addition to a reaction solution without isolation of RNA from a sample or prior heat treatment of the sample. A method for inspecting the presence or absence of a virus, comprising the following steps (1) to (3). (1) a step of mixing a sample and a reagent containing a polar organic solvent; and (2) a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, or a DNA polymerase having a reverse transcription activity, to the mixture. Step, (3) Step of performing a one-step RT-PCR reaction after sealing the reaction vessel.

Description

  The present invention relates to a method for detecting a virus by nucleic acid amplification. More specifically, by mixing a sample with a polar organic solvent without isolating nucleic acid from the sample or performing heat treatment, for example, by adding a reaction solution of real-time reverse transcription polymerase chain reaction (qRT-PCR), The present invention relates to detection of a non-enveloped RNA virus contained in a sample or an environmentally wiped sample. The present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

Nucleic acid amplification is a technology that amplifies several copies of a target nucleic acid to a level at which it can be visualized, that is, to hundreds of millions or more. It is also widely used in microbial tests and the like.
A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of primer to single-stranded DNA template, and (3) the above-mentioned primer using DNA polymerase. This is a method of amplifying a target nucleic acid in a sample by repeating the three steps of elongation as one cycle and repeating this cycle. Annealing and elongation may be performed in two steps at the same temperature.

  When analyzing RNA, reverse transcription (Reverse Transcription; RT) for converting template RNA into cDNA is performed as a pre-stage of this PCR. This is called RT-PCR. The RT-PCR includes (1) two-step RT-PCR in which RT and PCR are performed discontinuously, and (2) one-enzyme-based one-step RT in which RT and PCR are continuously performed using a single enzyme. -PCR, (3) Two-enzyme one-step RT-PCR in which RT and PCR are continuously performed using two kinds of enzymes, reverse transcriptase and DNA polymerase.

  Among the RT-PCR, in the genetic test, one-step RT-PCR is preferred in order to avoid the contamination due to the high processing ability and the opening and closing of the reaction vessel during the reaction. In the one-enzyme one-step RT-PCR, a DNA polymerase having a reverse transcription activity such as Tth DNA polymerase or Taq DNA polymerase is used. On the other hand, in two-enzyme one-step RT-PCR, at least two kinds of enzymes, a reverse transcriptase and a DNA polymerase, are used.

  Examples of the reverse transcriptase include reverse transcriptases derived from retroviruses or bacteria (for example, MMLV (Moloney Murine Leukemia Virus; Moloney murine leukemia virus) -RT (Reverse Transcriptase), AMV (Avian Myeloblastosis virus); -RT, HIV (Human Immunodeficiency Virus) -RT, RAV (Russ-associated virus) 2-RT, EIAV (Equine Infectious Anemia Virus) -RT, Carboxide thermogeno heromorgan heromyo generformo genrhomogenoformo germano generogeno generformo generogeno genomicor genomic romance generogeno genomicorman genomic romance generogeno genomicorman genomic romance generogeno hydrogen genermo genomic romancer genomic romance generogeno hydrogen gener omno gener romance gener omno gener s romance romance gener rom gen rom rom gen rom rom gen gen rom s rom s gen s s s gener ent gen s s s s s − − − ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ ＲＴ These mutants are used. Also, as a DNA polymerase. Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof are used.

  It is known that the one-enzyme one-step RT-PCR method has lower sensitivity than the two-step RT-PCR method (Non-Patent Document 1). In contrast, the two-enzyme one-step RT-PCR is more sensitive than the single enzyme-coupled RT-PCR because the reverse transcriptase activity of retrovirus-derived reverse transcriptase is higher than the reverse transcriptase activity of DNA polymerase. Is high (Non-Patent Document 2). However, although two-enzyme one-step RT-PCR has higher sensitivity than two-step RT-PCR, it has been reported that reverse transcriptase and DNA polymerase interfere with each other and reduce the reaction efficiency (Non-Patent Document 3). ). In order to suppress this interference, use of increasing the template RNA, non-homologous tRNA, T4 gene 32 protein, or increasing the ratio of DNA polymerase to reverse transcriptase has been used.

  For example, norovirus, one of the pathogenic RNA viruses, is a single-stranded RNA virus that causes acute gastroenteritis. It is a virus of high public health interest because it is highly infectious and causes mass food poisoning and outbreaks. Norovirus is classified into two gene groups, Genogroup I (G1) and Genogroup II (G2). In the examination of norovirus pathogens, a tissue culture method has not been established, and a method of detecting a viral gene using an electron microscopy method, an immunological antigen detection method by ELISA, or a nucleic acid amplification technique has been developed. Among these, the RT-PCR method based on the notification of the monitoring and safety section of the Safety Department of the Pharmaceutical and Food Bureau of the Ministry of Health, Labor and Welfare (Food Safety Inspector No. 1105001) has become widespread as an official method.

  The main cause of norovirus infection is eating and drinking foods contaminated with norovirus.However, since infections are often transmitted through human hands, regular stool tests are conducted at cooking facilities, medical sites, nursing homes, and nursery schools. Is required. The mass cooking facility hygiene management manual has added that the inspection of stool of cook workers and the like should include a norovirus test from October to March, which is the epidemic season of norovirus, if necessary. This is because there are quite a few people who are infected with the virus but have no symptoms (health carriers), and these people may spread the infection unknowingly. In addition, cooks who have symptoms such as diarrhea and vomiting should consult a medical institution and, if found to be infected with norovirus, conduct high-sensitivity tests such as real-time PCR and have no norovirus. It is desired that appropriate measures be taken, such as refraining from cooking operations that directly touch the food, until the condition is confirmed.

  Norovirus is a virus having no enveloping and having an icosahedral structure consisting of a capsid protein of about 30 nm. The viral RNA genome is encapsulated in this capsid structure. Therefore, conventionally, detection of norovirus from a stool sample is performed, for example, by preparing a 10% emulsion of stool, extracting and purifying RNA from a centrifuged supernatant using a commercially available virus RNA extraction kit, and using this RNA extract. Norovirus has been detected (Food Security Commissioner 1105001). However, in order to test a large number of samples in a short time, this RNA extraction operation is complicated. Therefore, in recent years, a method has been adopted in which the presence or absence of a virus is detected by destroying the capsid from a stool sample by heat treatment and subjecting the treatment solution exposing the viral RNA to RT-PCR (Non-Patent Document 4). . At this time, by omitting the extraction of RNA, a PCR reaction inhibitor such as a polysaccharide contained in the stool sample is brought in.

  Some measures have been taken to reduce these effects in pretreatment agents and PCR reaction solutions (Patent Document 1). It has been reported that stool PCR inhibition can be improved by the addition of betaine, BSA, T4 gene 32 protein, and a protease inhibitor. It was unclear whether detection by PCR could be improved (Non-Patent Document 5). The present inventors have developed a quaternary ammonium salt (betaine, L-carnitine, etc.) having a structure in which three methyl groups have been added to amino acids, bovine serum albumin, glycerol, glycol, and all or part of gelatin. It has been found that the combination of can eliminate PCR-induced inhibition of stool.

  On the other hand, since the heat treatment of the stool sample inactivates the reverse transcriptase, it is necessary to add an RT-PCR reaction solution after the heat treatment. Here, opening and closing of the reaction vessel and addition of the reagent require much labor and labor. In addition, there is the risk of contamination between samples. K. Kang et al. Report that highly pathogenic North American porcine reproductive and respiratory syndrome virus RNA can be detected by RT-PCR directly from porcine serum samples (Non-Patent Document 6). In this report, simply mixing a sample directly with an RT-PCR reaction solution and reacting disrupts the viral envelope structure and detects viral RNA. However, in a virus having no envelope capable of infecting the digestive tract, such as a norovirus, RNA is retained in a rigid capsid structure that is resistant to inactivation by gastric acid, surfactant activity of bile acid, and the like. Therefore, in some cases, the capsid structure is retained only by directly subjecting an untreated sample to RT-PCR, and it may not be possible to sufficiently detect RNA. Non-Patent Document 7 reports that the effect of destabilizing the capsid structure on a virus by a polar organic solvent varies depending on the type of virus, making it more difficult to detect RNA.

  Therefore, there is a method that can efficiently detect the presence or absence of viral RNA by adding a chaotropic agent to the RT-PCR reaction solution without previously isolating or heat-treating RNA from a stool sample or a concentrated sample for wiping test. However, it is known that addition of an excessive chaotropic agent inhibits the PCR reaction, and it has been difficult to shorten the PCR reaction time. Further, with the shortening of the PCR reaction time, there is a case where the detection sensitivity of norovirus is reduced even in the conventional heat treatment method. Thus, there is a need for a more efficient method for pretreating a viral RNA-containing specimen, which is less likely to inhibit the PCR reaction.

JP 2015-119656 A

BioTechniques, Vol. 17, 1994, pp. 1034-1036. BioTechniques, Vol. 25, 1998, pp. 230-234. Nucl. Acids Res. 20, Vol. 1992, pp. 1487-1490. J. Virol. Methods, 163, 2010, 282-286. J. Virol. Microbiol. 38, 2000, 4463-4470. J. Animal Science And Biotechnology, Vol. 5, 2014, p. 45. Bull, Natl. Inst. Health Sci. 129, 2011, pp. 37-54.

  An object of the present invention is to eliminate the presence or absence of viral RNA by one-step RT-PCR without the need for isolating non-enveloped virus RNA from a sample or prior heat treatment of the sample, with a precision higher than conventionally known techniques. Is to be detected.

In view of the above circumstances, the present inventors have conducted intensive studies and as a result, after mixing a sample that has not been subjected to viral RNA purification or prior heat treatment with a polar organic solvent, and subjecting it to one-step RT-PCR, It has been found that envelope virus RNA can be detected.
Furthermore, the present inventors determined the concentration of the polar organic solvent required for denaturation of the virus capsid structure and the concentration of the polar organic solvent allowed for the one-step RT-PCR reaction, and placed the polar organic solvent, the sample, After sequentially adding RT-PCR reaction solutions and sealing the reaction vessel, it was found that viral RNA could be detected only by performing a reaction in a temperature cycle for RT-PCR, and thus completed the present invention.

A typical invention of the present application is as follows.
Item 1. A method for testing an enveloped RNA virus in a sample, the method comprising the following steps without purifying the RNA from the sample or prior heat treatment of the sample, comprising the following steps: How to test for viruses to test.
(1) mixing a sample and a reagent containing a polar organic solvent,
(2) a step of adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, or a DNA polymerase having a reverse transcription activity, to the mixture;
(3) A step of performing a one-step RT-PCR reaction after sealing the reaction vessel.
Item 2. Item 3. The virus inspection method according to Item 1, wherein the steps (1) and (2) are performed in the same container.
Item 3. Item 3. The virus inspection method according to Item 1 or 2, wherein the one-step RT-PCR reaction is performed without opening and closing the lid even after the reaction vessel is sealed in the step (3).
Item 4. Item 4. The method for testing a virus according to any one of Items 1 to 3, wherein the sample is feces.
Item 5. Item 4. The virus inspection method according to any one of Items 1 to 3, wherein the sample is a suspension suspended in water, physiological saline, or a buffer.
Item 6. Item 4. The virus inspection method according to any one of Items 1 to 3, wherein the sample is a centrifugal supernatant of the suspension.
Item 7. Item 4. The method according to any one of Items 1 to 3, wherein the sample is a sample obtained by suspending an environmental wiping test sample in water, physiological saline or a buffer, and concentrating the suspension.
Item 8. Item 8. The method for testing a virus according to any one of Items 1 to 7, wherein the RNA virus having no envelope is a norovirus.
Item 9. Item 9. The virus inspection method according to Item 8, wherein it is determined whether the norovirus is a G1 type or a G2 type.
Item 10. The polar organic solvent is 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 10. The method for testing a virus according to any one of Items 1 to 9, wherein
Item 11. Item 11. The virus inspection method according to any one of Items 1 to 10, wherein the DNA polymerase is any one selected from the group consisting of Taq, Tth and mutants thereof.
Item 12. The origin of reverse transcriptase is any one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and mutants thereof. The method for testing a virus according to any one of the above.
Item 13. The one-step RT-PCR reaction solution in the step (3) comprises a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter, referred to as “betaine-like quaternary ammonium”). Item 13. The method for testing a virus according to any one of Items 1 to 12, comprising at least one selected from the group consisting of bovine serum albumin, glycerol, glycol, and gelatin at 5 mg / ml or more.
Item 14. Item 14. The method for testing a virus according to Item 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
Item 15. The method according to any one of claims 1 to 14, wherein the time required from the processing of the sample to the completion of the one-step RT-PCR reaction is within one hour.
Item 16. A kit for testing a virus without an envelope, comprising a reagent containing a polar organic solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution.
Item 17. Item wherein the one-step RT-PCR reaction solution in the step (3) contains at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin of 0.5 mg / ml or more, glycerol, glycol and gelatin. 17. The kit for testing a virus according to 16 above.
Item 18. Item 18. The virus testing kit according to Item 16 or 17, further comprising a primer pair corresponding to a detection region of an RNA virus to be detected.
Item 19. Item 19. The virus test kit according to any one of Items 16 to 18, further comprising a hybridization probe corresponding to a detection region of an RNA virus to be detected.
Item 20. Item 20. The virus testing kit according to any one of Items 16 to 19, wherein the RNA virus having no envelope is a norovirus.
Item 21. Item 21. The virus testing kit according to Item 20, wherein it is determined whether the norovirus is a G1 type or a G2 type.

  According to the present invention, isolation of a viral RNA from a sample or heat treatment of the sample in advance is not required, and the sample is mixed with a polar organic solvent and then added to a one-step RT-PCR reaction solution, so that norovirus in the sample can be obtained. It is possible to detect the presence or absence of any non-enveloped virus. As a result, the inspection work becomes more efficient, so that the test amount of a subject who has no symptoms even if the virus is infected can be increased, which also contributes to the prevention of infectious diseases. Further, the opening and closing operation of the lid of the reaction vessel is also omitted by omitting the heat treatment step. As a result, the risk of scattering of the virus-containing sample when opening and closing the lid can be eliminated, and the risk of contamination of other samples can be reduced. As a result, the risk of occurrence of false positives can be suppressed, and the accuracy of the inspection work can be further increased.

It is a figure which shows the result of having examined the PCR reaction inhibitory effect in each solvent. FIG. 9 is a view showing the results of examining the effect of sample pretreatment in each solvent. It is a figure which shows the result of having examined the comparison of each pre-processing method at the time of reaction time shortening. It is a figure which shows the working process of each pre-processing method. It is a figure which shows the result of having examined the comparison of the working time of each pre-processing method.

  Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

  One embodiment of the present invention is a test for a non-enveloped RNA virus such as a norovirus in a sample, in which a sample and a reagent containing a polar organic solvent are mixed without performing purification of viral RNA from the sample or prior heat treatment of the sample. And a method for testing for the presence or absence of a virus, which comprises adding a one-step RT-PCR reagent containing a reverse transcriptase and a DNA polymerase, or a DNA polymerase having a reverse transcription activity.

The method for examining the presence or absence of a virus in a sample of the present invention is a method for examining the presence or absence of a virus, which comprises at least the following steps.
(1) mixing a sample and a reagent containing a polar organic solvent,
(2) a step of adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, or a DNA polymerase having a reverse transcription activity, to the mixture;
(3) A step of performing a one-step RT-PCR reaction after sealing the reaction vessel.
The steps (1) and (2) are preferably performed in the same container. That is, it is preferable not to transfer all or part of the mixed solution to another container between steps (1) and (2). In step (1), the entire amount of the mixed solution may be supplied to step (2), or a part of the mixture may be transferred to another container to perform step (2). Furthermore, in the step (3), it is preferable that the lid of the reaction vessel is not opened and closed after the reaction vessel is sealed.

  The RNA virus to be tested in the present invention is not particularly limited as long as it is a non-enveloped RNA virus having no envelope derived from a lipid bilayer. Such non-enveloped RNA viruses include Astroviridae Astrovirus, Caliciviridae Sapovirus, Norovirus, Picornaviridae Hepatitis A Virus, Echovirus, Enterovirus, Coxsackivirus, Poliovirus, Rhinovirus, Examples include hepatitis E virus of the viridae family, rotavirus of the reoviridae family, but are not particularly limited, and are particularly useful for detecting norovirus. Many non-enveloped viruses can infect the gastrointestinal tract by fecal-oral infection and the like, and RNA is retained in a rigid capsid structure that is resistant to inactivation by gastric acid, surfactant activity of bile acid, and the like. For this reason, non-enveloped viruses may not be inactivated even under conditions in which enveloped viruses (such as influenza viruses) are inactivated.

  Examples of the sample used in the present invention include feces (excrementary stool, rectal stool), vomit, and saliva, but are not particularly limited, and can be used for all samples derived from living organisms. In particular, it is useful for detection from faeces (excretion, rectal stool). The stool is characterized in that it contains proteases and nucleases as contaminants, as well as a large amount of Escherichia coli-derived proteins and nucleic acids. It is known that the reaction solution components such as enzymes, primers, and nucleic acid probes used in the RT-PCR reaction are digested or inactivated due to the influence of impurities contained in feces, resulting in reduced detection sensitivity. The present invention is characterized in that there is no need to isolate RNA from these samples using a commercially available RNA purification kit or to perform a prior heat treatment for exposing the RNA from the virus structure by heat treatment. The sample may be directly subjected to detection, or in order to reduce the influence of contaminants on the reaction and obtain a more stable test result, use a sample in which the sample is suspended in water, saline, or a buffer. There may be. Furthermore, in the case of a sample containing many impurities such as feces, centrifugation may be performed and the supernatant may be used. Alternatively, filter filtration may be performed. Examples of the buffer include, but are not particularly limited to, Hanks buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and tricine buffer.

  A sample according to another aspect of the present invention is a wiping test sample. A wiping test is useful for elucidating the contamination route and grasping the contamination status such as the facility environment. In the present invention, the wiping test is not particularly limited, but for example, a sample obtained by wiping a corresponding section or facility with a cotton swab or the like, eluting with water or a buffer, and concentrating with polyethylene glycol (PEG) precipitate or the like. . Specific examples of the wiping test include “Improvement of Norovirus Testing Method for Wiping Specimen” (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). There is no particular limitation, and a wide range of methods according to this is included. Examples of wiping parts include kitchen utensils such as cutting boards, kitchen knives, towels, dishes, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathrooms and other faucets, cook hands and fingers, and bathrooms. Facilities such as toilets, toilets, handrails, living rooms, and the like. Although not a wiping test, it can also be applied to a concentrated sample of a sewage sample as an environmental test.

  In the present invention, polarity refers to an electronic bias existing in a molecule, and a molecule in which the center of gravity of a positive charge and the center of a negative charge in a molecule do not match is called a polar molecule. A solvent composed of polar molecules is called a polar solvent. By using a polar organic solvent composed of an organic compound among polar solvents, it is possible to destabilize a higher-order structure of a biomolecule such as a nucleic acid or a protein. By utilizing this property, the effect of weakening the hydrophobic bond and the like of the capsid protein of the virus and destabilizing the capsid structure is exhibited. As described above, it is known that the effect of destabilizing the capsid structure on a virus by a polar organic solvent varies depending on the type of virus (Non-Patent Document 7). This is thought to be due to differences in the strength of hydrophobic binding and the like due to differences in the properties of the capsid proteins possessed by the virus.

  As the polar organic solvent, specifically, ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethylsulfoxide, acetone, acetonitrile, ethanol , Methanol, 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Preferably, methanol, triethylamine, dimethyl sulfoxide, and acetone are used. Further, a mixed solution containing two or more of these polar organic solvents may be used. The lower limit concentration of the polar organic solvent as a denaturant for the capsid protein is not particularly limited as long as the concentration is such that the capsid protein is denatured, although it depends on the type of the polar organic solvent and other additives. In addition, since the capsid protein differs depending on the type of virus, the effective concentration of the polar solvent differs for each virus, but the effective concentration of the polar solvent relative to the sample amount is usually 10% or more and less than 100%, more preferably 30% or less. % To 90%, more preferably 50% to 85%.

  The polar organic solvent may be used in combination with one or more surfactants, reducing agents, chelating agents, metal salts,

  Said polar organic solvents are usually also known as inhibitors of PCR. Therefore, among the polar organic solvents, those having a small difference between the concentration required for protein denaturation and the allowable concentration for carry-in to PCR are selected, so that the polar organic solvent, the sample, and the one-step RT-PCR reaction solution are sequentially added. By doing so, the detection operation can be easily performed in the same container from the denaturation of the capsid protein to the one-step RT-PCR reaction without opening and closing the container in the middle. An example of such a polar organic solvent is particularly preferably dimethyl sulfoxide. For example, when 1 μL of dimethyl sulfoxide and 1 μL of a sample are mixed and 48 μL of a one-step RT-PCR solution is added, the concentration of dimethyl sulfoxide brought into the reaction solution is 2%. 2% dimethyl sulfoxide is a concentration that is acceptable even when the RT-PCR solution is brought in.

  During the sample transfer or heat treatment step, the operation of opening and closing the reaction vessel occurs. The opening and closing operation of the reaction vessel becomes complicated and causes an increase in operation time. In addition, opening and closing the reaction container containing the virus-containing sample poses a risk of scattering of the virus and virus-derived RNA. The spread of the virus is a threat to the safety and health of workers, and at the same time, contaminates the inspection work environment. Since the scattered RNA virus is aerosolized in the workplace, there is a risk of contaminating other samples being tested at the same time. For this reason, the method of testing for the presence or absence of a virus using RT-PCR without a lid opening / closing step is more than a simple operation.

  The one-step RT-PCR solution added to the mixture contains reverse transcriptase and DNA polymerase. It is preferable to use Tth DNA polymerase, Taq DNA polymerase, or the like, which is a DNA polymerase having reverse transcriptase activity. More preferably, two types of enzymes are used, and at least two types of enzymes, a reverse transcriptase and a DNA polymerase, are used.

  The origin of the reverse transcriptase contained in the one-step RT-PCR reaction solution is not particularly limited as long as RNA can be converted to DNA. However, MMLV (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV -RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenogenforman DNA polymerase) and variants thereof. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.

  Examples of the DNA polymerase contained in the one-step RT-PCR reaction solution include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. Is not particularly limited. More preferably, use of Taq, Tth or a mutant thereof is used. Particularly preferred is the use of Tth or a variant thereof. Furthermore, in order to enhance the effect of suppressing non-specific reactions, the enzyme activity of the DNA polymerase during the reverse transcription reaction can be increased by using the DNA polymerase in combination with an anti-DNA polymerase antibody or by introducing the thermolabile blocking group into the DNA polymerase by chemical modification. Is preferably suppressed and application to hot start PCR is possible.

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

  The buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, Bicine, and the like. The pH is adjusted to 6 to 9, more preferably 7 to 8, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like. The concentration of the buffer added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added in order to set 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.

  As the dNTP used in the present invention, dATP, dCTP, dGTP, and dTTP are each added in an amount of 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 the magnesium salt include magnesium chloride, magnesium sulfate, and magnesium acetate, and examples of the manganese salt include manganese chloride, manganese sulfate, and manganese acetate.

  Further, as an additive contained in the one-step RT-PCR reaction solution, a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter, referred to as “betaine-like quaternary ammonium”), It preferably contains at least 0.5 mg / ml of at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.

  Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine. A quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid, There is no particular limitation. The structure of the betaine-like quaternary ammonium salt is a compound having stable positive and negative charges in the molecule, exhibits properties like a surfactant, and is considered to cause instability of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase. The preferred betaine-like quaternary ammonium salt concentration is 0.1M to 2M, more preferably 0.2M to 1.2M.

  Bovine serum albumin contained in the one-step RT-PCR reaction solution is preferably at least 0.5 mg / ml or more, more preferably at least 1 mg / ml or more. In a sample with many contaminants, the concentration of bovine serum albumin is preferably 2 mg / ml or more, more preferably 3 mg / mg or more, and good detection becomes possible.

  The gelatin contained in the one-step RT-PCR reaction solution is considered to be derived from skin, bones, tendons, or fish scales and skins of animals such as cows and pigs, and to contribute to the stabilization of PCR enzymes. The concentration used is preferably such that the PCR amplification is stabilized while the fluorescence detection is not hindered. Preferably it is 1 to 5%, more preferably 1 to 2%. The origin of gelatin is not particularly limited, but those derived from fish are more preferable than those derived from cattle or pig, because they have lower jelly strength and handle the reaction solution better.

  Furthermore, they can be used in combination with substances known in the art to promote RT-PCR. Accelerators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur or acetate containing compounds, dimethyl sulfoxide (DMSO), 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-100), Triton X-114 (Triton X-114) , Tween 20 (Tween20), Nonidet P40, although Briji58 and the like, without limitation. In order to further reduce reaction inhibition, ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane- A chelating agent such as N, N, N ', N'-tetraacetic acid (BAPTA) may be included.

  The primer pair used in the present invention is a pair of primers corresponding to the detection region of the RNA virus to be detected, and one pair of primers in which one primer is complementary to the DNA extension product of the other primer. No. Another embodiment is a so-called multiplex PCR in which two or more pairs of the above primers are contained. Further, when the target nucleic acid is of a subtype, it may contain a degenerate primer. In the present invention, when detecting norovirus, which is a kind of RNA virus having no envelope, as an example of a primer pair, as a primer for detecting norovirus, a notification from the Monitoring and Safety Division of the Safety Department of the Pharmaceutical and Food Bureau of the Ministry of Health, Labor and Welfare (food safety) Primer No. 1105001), but not limited thereto.

  In another aspect, the present invention is a detection method further comprising at least one kind of a labeled hybridization probe or a double-stranded DNA-binding fluorescent compound. As a result, the analysis of the amplification product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis effort is reduced. Furthermore, there is no need to open the reaction vessel, and the risk of contamination can be reduced. By labeling each hybridization probe corresponding to a virus subtype with a different fluorescent dye, it is also possible to identify the virus subtype.

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

  As a hybridization probe used in the present invention, for example, a TaqMan hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972) US Pat. No. 5,804,375), molecular beacon (US Pat. No. 5,118,801), FRET hybridization probe (WO 97/46707, WO 97/46712). And WO 97/46714 pamphlet). Examples of the nucleotide sequence of a probe for detecting norovirus include, but are not limited to, the sequence described in the notification of the monitoring and safety section of the Safety Department of the Pharmaceutical and Food Bureau of the Ministry of Health, Labor and Welfare (Food Safety Inspection No. 1105001). Furthermore, when the target nucleic acid is of a subtype, it may contain a degenerate sequence.

  Another embodiment of the present invention is a kit for testing viral RNA in a sample, comprising a chaotropic agent, a reverse transcriptase and a DNA polymerase (or a DNA polymerase having reverse transcription activity), and a one-step RT-PCR reaction solution. This is a kit for testing an RNA virus without an envelope, characterized by comprising: The virus test kit of the present invention includes a reagent containing at least a polar organic solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. The one-step RT-PCR reaction solution preferably contains at least one of betaine-like quaternary ammonium salt, 0.5 mg / ml or more bovine serum albumin, glycerol, glycol and gelatin. It is preferable to include a primer pair corresponding to the detection region of the RNA virus to be detected, and a hybridization probe corresponding to the detection region of the RNA virus to be detected.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

Embodiment 1 FIG. Screening of Samples Required for Heat Treatment (1) RT-PCR Reaction Solution The reaction solution having the following composition was used as a basic composition, and the influence of an organic solvent was evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Pretreatment liquid 4μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Sample preparation method Fifteen samples of norovirus G2-positive human feces were suspended in 0.5 ml of sterilized water so as to be 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.

(3) Reaction Under the conditions for performing the pretreatment, 1 μL of each sample prepared in 4 μL of the pretreatment solution was added. The mixed solution with the pretreatment solution was subjected to a heat treatment at 85 ° C. for 1 minute using a thermal cycler (Gene Amp (registered trademark) PCR System 9700) manufactured by Applied Bioscience. To the pre-treated solution after the heat treatment, 45 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution) was added to obtain a PCR reaction solution. Under the condition that the pre-treatment was not performed, 4 μL of the pre-treatment solution was added to 45 μL of the RT-PCR reaction solution, and 1 μL of each prepared sample was added. Using the CFX96WELL DEEP manufactured by BioRad, a real-time PCR reaction was performed at the following temperature cycle. Fluorescence readings were taken during the second 30 cycles of the 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C for 1 second-54 ° C for 22 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 22 seconds 30 cycles (PCR)

(4) Results In the probe solution (norovirus detection kit G1 / G2-high-speed probe detection-attached to Toyobo), an internal control gene is detected in the FAM channel, norovirus G1 in the Cy5 channel, and norovirus G2 in the ROX channel. Here, the Cq values of G1 and G2 RNA are shown. As a result, under the conditions of the heat treatment, detection of all G2-positive samples was confirmed (Table 1). On the other hand, among the samples that were not subjected to the pretreatment, the detection was not confirmed in 11 samples (Table 2). This is considered to be due to the fact that the capsid structure of the virus in the sample was not broken because the heat treatment was not performed, and no RNA molecules came out of the virus particles. In this study, a sample for which a G2 positive signal could not be confirmed was regarded as a sample requiring heat treatment.

Example 2.1 Screening of Solvent Usable for Step RT-PCR Reaction (1) RT-PCR Reaction Solution The reaction solution having the following composition was used as a basic composition, and the influence of each solvent on RT-PCR was evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Pretreatment liquid 4μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Solvent condition 1 distilled water condition 2 8M urea condition 3 ethanol condition 4 methanol condition 5 1-propanol condition 6 2-propanol condition 7 1-butanol condition 8 acetone condition 9 1,4-dioxane condition 10 pyridine condition 11 dimethyl Sulfoxide condition 12 Triethylamine condition 13-3-Methylbutyl acetate condition 14 Anisole condition 15 Hexane condition 16 Benzine

(3) Reaction 2 μL of the solvent was added to each of the reaction tubes, and 49 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution + pretreatment solution) was added. To this was added 250 copies of Norovirus G2 synthetic RNA. Using the CFX96WELL DEEP manufactured by BioRad, a real-time PCR reaction was performed at the following temperature cycle. Fluorescence readings were taken during the second 30 cycles of the 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C for 1 second-54 ° C for 22 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 22 seconds 30 cycles (PCR)

(4) Results FIG. 1 shows the Cq values of G2 RNA obtained as a result. As a result, 1-propanol, 2-propanol, 1-butanol, 1,4-dioxane, and pyridine completely inhibited the RT-PCR reaction, and no G2 signal was confirmed. In addition, in the case of anisole, the Cq value was significantly increased as compared with the condition of adding distilled water, and it was confirmed that PCR inhibition occurred.

Embodiment 3 FIG. Screening of Solvent Having Pretreatment Effect on Virus-Containing Sample (1) RT-PCR Reaction Solution The reaction solution having the following composition was used as a basic composition, and the pretreatment effect of each solvent on the sample was evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Pretreatment liquid 4μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Solvent conditions Condition 1 Distilled water Condition 2 8M urea Condition 3 Ethanol condition 4 Methanol condition 5 Acetone condition 6 Dimethyl sulfoxide condition 7 Triethylamine condition 8 Acetic acid-3-methylbutyl condition 9 Anisole condition 10 Hexane condition 11 Benzine

(3) Sample preparation method Norovirus G2 positive human stool specimen a1, which is a specimen requiring heat treatment, was suspended in 0.5 ml of sterilized water so as to be 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.

(4) Reaction 2 μL of each of the above solvents was added to the reaction tube, and it was confirmed that the solvent had fallen to the bottom of the tube. 1 μL of the prepared sample was added and mixed with each solvent. To this, 49 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution + pretreatment solution) was added. Using the CFX96WELL DEEP manufactured by BioRad, a real-time PCR reaction was performed at the following temperature cycle. Fluorescence readings were taken during the second 30 cycles of the 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C for 1 second-54 ° C for 22 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 22 seconds 30 cycles (PCR)

(5) Result FIG. 2 shows the Cq value of the G2 RNA obtained as a result. As a result, no detection was confirmed for ethanol, 3-methylbutyl acetate, anisole, hexane, and benzine, and no pretreatment effect on the virus-containing specimen was confirmed. In contrast, the detection of G2 signal was confirmed for methanol, acetone, triethylamine, and dimethyl sulfoxide. In particular, dimethylsulfoxide has a Cq value of at least 3 lower than urea having a pretreatment effect, confirming that the pretreatment effect on the virus-containing specimen is high.

Embodiment 4. FIG. Examination of Effective Concentration of Dimethyl Sulfoxide (1) RT-PCR Reaction Solution The reaction solution having the following composition was used as a basic composition, and the effective concentration of dimethyl sulfoxide for the pretreatment effect on the virus-containing specimen was evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Pretreatment liquid 4μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Sample preparation method Norovirus G2-positive human stool specimens e1, f1, and h2, which are specimens requiring heat treatment, were suspended in 0.5 ml of sterilized water so as to be 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.

(3) Mixing method with dimethyl sulfoxide A 100%, 90%, 80%, 70%, 60%, 50%, 40% dimethyl sulfoxide aqueous solution was prepared with distilled water, and samples e1, f1, h2 were prepared under the following conditions. And mixed.
Condition 1 1 μL of each sample, 2 μL of 100% dimethyl sulfoxide (effective concentration 67%)
Condition 2 1 μL of each sample, 2 μL of 90% dimethyl sulfoxide (effective concentration 60%)
Condition 3 1 μL of each sample, 2 μL of 80% dimethyl sulfoxide (effective concentration 53%)
Condition 4 1 μL of each sample, 2 μL of 70% dimethyl sulfoxide (effective concentration 47%)
Condition 5: 1 μL of each sample, 2 μL of 60% dimethyl sulfoxide (effective concentration: 40%)
Condition 6: 1 μL of each sample, 2 μL of 50% dimethyl sulfoxide (effective concentration: 33%)
Condition 7: 1 μL of each sample, 2 μL of 40% dimethyl sulfoxide (effective concentration: 27%)
Condition 8: 1 μL of each sample, 2 μL of distilled water

(4) Reaction 2 μL of each aqueous dimethyl sulfoxide solution under the above conditions was added to the reaction tube, and it was confirmed that the solution had fallen to the bottom of the tube. 1 μL of the prepared sample was added and mixed. To this, 49 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution + pretreatment solution) was added. Using the CFX96WELL DEEP manufactured by BioRad, a real-time PCR reaction was performed at the following temperature cycle. Fluorescence readings were taken during the second 30 cycles of the 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C for 1 second-54 ° C for 22 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 22 seconds 30 cycles (PCR)

(5) Result As a result, Table 3 shows the Cq value of the G2 signal under each condition. Although the effective concentration varies depending on the sample, the effective concentration of dimethylsulfoxide in the mixture of the sample and dimethylsulfoxide is 40% or more, more preferably 50% or more. Admitted. At this time, the concentration of dimethyl sulfoxide in the RT-PCR reaction solution is 2.4% to 4%.

Embodiment 5 FIG. Examination of allowable amount of dimethyl sulfoxide to be brought into RT-PCR reaction solution (1) RT-PCR reaction solution The reaction solution having the composition shown below was used as a basic composition, and the effect of the amount of dimethyl sulfoxide carried on RT-PCR was evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Addition amount of dimethyl sulfoxide Condition 1 0 μL dimethyl sulfoxide, 5 μL distilled water (final concentration 0%)
Condition 2 1 μL dimethyl sulfoxide, 4 μL distilled water (final concentration 2%)
Condition 3 2 μL dimethyl sulfoxide, 3 μL distilled water (final concentration 4%)
Condition 4 3 μL dimethyl sulfoxide, 2 μL distilled water (final concentration 6%)
Condition 5 4 μL dimethyl sulfoxide, 1 μL distilled water (final concentration 8%)
Condition 6 5 μL dimethyl sulfoxide, 0 μL distilled water (final concentration 10%)

(3) Reaction Dimethyl sulfoxide and distilled water were added to a reaction tube by the volume described in the above conditions, and 45 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution) was added. To this, 250 copies, 50 copies, and 10 copies of synthetic RNAs of Norovirus G2 and G1 were added. Using the CFX96WELL DEEP manufactured by BioRad, a real-time PCR reaction was performed at the following temperature cycle. Fluorescence readings were taken during the second 30 cycles of the 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C for 1 second-54 ° C for 22 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 22 seconds 30 cycles (PCR)

(4) Results As a result, Table 4 shows the Cq values of the G1 and G2 signals of each sample. When the final concentration of dimethyl sulfoxide was 10%, the G1 and G2 signals showed an increase in the Cq value at 250 copies, and no signal was observed at 50 copies and 10 copies. From these results, when carrying out RT-PCR for the purpose of detecting about 50 copies, the allowable amount of dimethyl sulfoxide carried into the reaction solution is considered to be 10% or less, more preferably 8% or less. However, when detecting norovirus G1 and G2 RNA of 250 copies or more, carry-in of 10% or more is allowed.

Embodiment 6 FIG. Comparison between dimethylsulfoxide treatment and conventional pretreatment method (1) RT-PCR reaction solution Pretreatment of virus-containing specimen with dimethylsulfoxide under the condition that the reaction solution having the following composition is used as the basic composition and the PCR reaction time is shortened The effect and the conventional pretreatment method were compared and evaluated.
Reaction solution 30 μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Primer solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Probe solution 5μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)
Enzyme solution 5μL
0.8 unit / μL rTth mutant DNA polymerase
0.24 μg / μL anti-Tth antibody (Toyobo)
1 unit / μl Reversetrace (Toyobo)
Pretreatment liquid 4μL
(Norovirus detection kit G1 / G2-high-speed probe detection-(Toyobo) accessory)

(2) Sample preparation method Norovirus G2-positive human stool specimens e2 and g2, which are required to be subjected to heat treatment, were suspended in 0.5 ml of sterilized water so as to be 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.

(3) Detection by dimethyl sulfoxide treatment After mixing 2 μL of dimethyl sulfoxide and 1 μL of the sample, 45 μL of the above RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution) was added, and BioRad CFX96WELL DEEP was used. The real-time PCR reaction was performed at the temperature cycle described in (6), and the fluorescent value was read in the extension step of 54 ° C. in the latter 30 cycles.

(4) Detection by chaotropic treatment After mixing 2 μL of an 8M urea aqueous solution and 1 μL of a sample, 45 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution) was added, and CFX96WELL manufactured by BioRad was added.
Using DEEP, the real-time PCR reaction was performed at the temperature cycle described in (6), and the fluorescence value was read in the extension step of 54 ° C. in the latter 30 cycles.

(5) Detection by Heat Treatment 4 μL of the pretreatment solution was added to 1 μL of the sample, and heat treatment was performed at 85 ° C. for 1 minute. After mixing 45 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution), a real-time PCR reaction was performed using the CFX96WELL DEEP manufactured by BioRad at the temperature cycle described in (6). Fluorescence readings were taken in 30 cycles of the 54 ° C. extension step.

(6) Reaction cycle 42 ° C. for 3 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 5 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 5 seconds 30 cycles (PCR)

(7) Results The Cq value of the G2 signal when each pretreatment method was performed is shown in FIG. In the present study, the pretreatment method from which a G2 signal was obtained was only treatment with dimethyl sulfoxide. This is because the RT-PCR reaction time is shortened, the efficiency of disruption of the capsid structure of the virus is low, and under the condition that the leakage of RNA into the reaction solution is small, the amount of RNA serving as a template is insufficient, and RT-PCR is not performed. The cause may be that they did not respond sufficiently. Therefore, it was shown that the capsid crushing efficiency of the virus in the specimen was higher in the dimethyl sulfoxide treatment method of the present invention than in the conventionally known heat treatment method and chaotropic agent treatment method.

Embodiment 7 FIG. Comparison of Working Time between Dimethyl Sulfoxide Treatment and Conventional Heat Treatment Method (1) Working Steps The main working steps of the pretreatment method including the dimethyl sulfoxide treatment and the heating treatment step are shown in FIG. A special jig (Fast Gene Cap easy) of Nippon Genetics Co., Ltd. was used for opening and closing the lid of the reaction vessel in the method including the heat treatment step.

(2) Comparative method In the work flow shown in FIG. 4, the time required for preparing 96 samples of the RT-PCR reaction was measured. The measurement was performed five times, and the average value was calculated and compared.

(3) Result The conventional method including the heat treatment step requires about 21 minutes and 30 seconds, whereas the dimethyl sulfoxide treatment of the present invention requires about 14 minutes, resulting in a 36% reduction in working time. (FIG. 5).
RT-PCR reaction time is 45 minutes when the reaction is performed in the following reaction cycle using CFX96WELL DEEP manufactured by BioRad.
42 ° C for 3 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 5 seconds 10 cycles (PCR)
95 ° C for 1 second-54 ° C for 5 seconds 30 cycles (PCR)
The total time required for the pretreatment method using the dimethyl sulfoxide treatment is 59 minutes, and the time required from the sample treatment to the measurement can be achieved within one hour.

  INDUSTRIAL APPLICABILITY The present invention is suitably used in molecular biology research, as well as in clinical tests and tests for food hygiene management.

Claims (21)

A method for testing an enveloped RNA virus in a sample, the method comprising the following steps without purifying the RNA from the sample or prior heat treatment of the sample, comprising the following steps: How to test for viruses to test.
(1) mixing a sample and a reagent containing a polar organic solvent,
(2) a step of adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a DNA polymerase, or a DNA polymerase having a reverse transcription activity, to the mixture;
(3) A step of performing a one-step RT-PCR reaction after sealing the reaction vessel. The virus inspection method according to claim 1, wherein the steps (1) and (2) are performed in the same container. The method according to claim 1 or 2, wherein in the step (3), after sealing the reaction container, a one-step RT-PCR reaction is carried out without opening and closing the lid even once. The method for testing a virus according to any one of claims 1 to 3, wherein the sample is feces. 4. The method according to claim 1, wherein the sample is a suspension suspended in water, physiological saline, or a buffer. The method for testing a virus according to any one of claims 1 to 3, wherein the sample is a centrifugal supernatant of the suspension. The virus according to any one of claims 1 to 3, wherein the sample is a sample obtained by suspending an environmental swab test sample in water, physiological saline or a buffer, and concentrating the suspension. Inspection methods. The method for testing a virus according to any one of claims 1 to 7, wherein the RNA virus having no envelope is a norovirus. The method according to claim 8, wherein it is determined whether the norovirus is a G1 type or a G2 type. The polar organic solvent is 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 The method for testing a virus according to any one of claims 1 to 9, wherein the method is any one of: The method for testing a virus according to any one of claims 1 to 10, wherein the DNA polymerase is any one selected from the group consisting of Taq, Tth and mutants thereof. 12. The reverse transcriptase is derived from one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and mutants thereof. The method for testing a virus according to any one of the above. The one-step RT-PCR reaction solution in the step (3) comprises a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter, referred to as “betaine-like quaternary ammonium”). The method according to any one of claims 1 to 12, comprising at least one selected from the group consisting of bovine serum albumin, glycerol, glycol, and gelatin at 5 mg / ml or more. The virus inspection method according to claim 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. The method according to any one of claims 1 to 14, wherein the time required from the processing of the sample to the completion of the one-step RT-PCR reaction is within one hour. A kit for testing a virus without an envelope, comprising a reagent containing a polar organic solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution. The one-step RT-PCR reaction solution in the step (3) contains at least one selected from the group consisting of betaine-like quaternary ammonium salts, bovine serum albumin of 0.5 mg / ml or more, glycerol, glycol and gelatin. Item 17. A kit for testing a virus according to Item 16. The virus test kit according to claim 16 or 17, further comprising a primer pair corresponding to a detection region of an RNA virus to be detected. The virus testing kit according to any one of claims 16 to 18, further comprising a hybridization probe corresponding to a detection region of an RNA virus to be detected. The virus testing kit according to any one of claims 16 to 19, wherein the RNA virus having no envelope is a norovirus. The test virus kit according to claim 20, wherein it is determined whether the norovirus is a G1 type or a G2 type.