WO2018198682A1 - Virus test method and virus test kit - Google Patents

Abstract

Provided are a method and a kit with which it is possible to detect the presence of RNA of a non-enveloped virus by addition to a reaction solution and one-step RT-PCR without isolation of the RNA from a sample or advance heat treatment of the sample. A method for testing whether or not a virus is present, the method comprising the following steps (1) to (3). (1) A step for mixing a sample and a reagent that contains a polar organic solvent. (2) A step for adding a one-step RT-PCR reaction solution that contains reverse transferase and DNA polymerase, or DNA polymerase having reverse transcription activity, to the mixed solution. (3) A step for performing a one-step RT-PCR reaction after sealing the reaction vessel.

Description

Virus inspection method and virus inspection kit

The present invention relates to a virus detection method by nucleic acid amplification. More specifically, the sample is mixed with a polar organic solvent without isolation of nucleic acid from the sample or heat treatment, and then a real-time reverse transcription polymerase chain reaction (qRT-PCR) reaction solution is added. The present invention relates to detection of an RNA virus having no envelope, which is contained in a sample or an environmental wipe sample. The present invention can also 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 that can be visualized, that is, several hundred million copies or more. It is also widely used in the microbiological examination of the
A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primer to template single-stranded DNA, (3) the primer using DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of extension as one cycle. In some cases, annealing and elongation are performed at the same temperature in two steps.

When analyzing RNA, reverse transcription (RT) for converting template RNA into cDNA is performed as a pre-stage of this PCR. This is called RT-PCR. This RT-PCR includes (1) two-step RT-PCR in which RT and PCR are discontinuously performed, and (2) one enzyme system in which RT and PCR are continuously performed using a single enzyme, one-step RT -PCR, (3) Roughly divided into three types, two-enzyme system, one-step RT-PCR, in which RT and PCR are continuously performed using two types of enzymes, reverse transcriptase and DNA polymerase.

Among the RT-PCR, the one-step RT-PCR is preferred for genetic testing in order to avoid high contamination and contamination due to opening and closing of the reaction vessel during the reaction. In the one-enzyme system 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 the two-enzyme one-step RT-PCR, at least two kinds of enzymes, reverse transcriptase and DNA polymerase, are used.

As the reverse transcriptase, a retrovirus derived from a retrovirus or a bacterium (for example, MMLV (Moloney Murine Leukemia Virus; Moloney Murine Leukemia Virus) -RT (Reverse Transscriptase), AMV (Avian Myeloblastosis virus blast virus) -RT, HIV (Human Immunodefense Virus)-RT, RAV (Rous-associated virus) 2-RT, EIAV (Equine Infectious Anemia Viral) -RT, Carboxido-thermas-Hydrogenous form Ze, etc.) and these variants are available. 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 system one-step RT-PCR method has lower sensitivity than the two-step RT-PCR (Non-patent Document 1). In contrast, two-enzyme one-step RT-PCR is more sensitive than 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 the two-enzyme system one-step RT-PCR has higher sensitivity than the two-step RT-PCR, it has been reported that reverse transcriptase and DNA polymerase interfere with each other to reduce reaction efficiency (Non-patent Document 3). ). In order to suppress this interference, use of an increase in template RNA, non-homolog tRNA, T4 gene 32 protein, or an increase in the ratio of DNA polymerase to reverse transcriptase has been performed.

For example, Norovirus, which is one of pathogenic RNA viruses, is a single-stranded RNA virus that causes acute gastroenteritis. It is a virus with high public interest because it is highly infectious and causes mass food poisoning and mass infection. Noroviruses are classified into two gene groups: Genogroup I (G1) and Genogroup II (G2). In norovirus pathogen testing, tissue culture methods have not been established, and methods for detecting viral genes using electron microscopy, ELISA for immunological antigen detection, or nucleic acid amplification techniques have been developed. Among them, the RT-PCR method based on the notice (safety supervisor 1105001) of the Ministry of Health, Labor and Welfare, Pharmaceutical Food Bureau, Safety Department, Safety Division is widely used as an official method.

It is mainly due to eating and drinking foods contaminated with norovirus as the cause of norovirus infection, but because there are many infections through human hands, regular stool inspections at cooking facilities, medical sites, elderly care facilities and nurseries etc. Is required. In the mass cooking facility hygiene management manual, it is added to the stool examination of cooks and the like that the examination of norovirus is included from October to March, which is the epidemic period of norovirus, as necessary. This is because there are not a few people (health carriers) who are infected with the virus but have no symptoms, and these people may unknowingly spread the infection. In addition, cooks with symptoms such as diarrhea and vomiting should visit a medical institution, and if they are found to be infected with norovirus, perform high-sensitivity tests such as real-time PCR and do not have norovirus It is desired to take appropriate measures such as refraining from the cooking work that directly touches food until it is confirmed.

Norovirus is a virus with an icosahedral structure consisting of a capsid protein of about 30 nm without an envelope. The viral RNA genome is encapsulated in this capsid structure. Therefore, conventionally, norovirus is detected from a stool sample by, for example, preparing a 10% emulsion of stool, extracting and purifying RNA from a centrifugal supernatant using a commercially available viral RNA extraction kit, and using this RNA extract. Norovirus has been detected (Food Safety Supervisor 1105001). However, this RNA extraction operation is complicated in order to examine a large number of specimens in a short time. 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 exposed to viral RNA to RT-PCR (Non-Patent Document 4). . At this time, by omitting RNA extraction, a PCR reaction inhibiting substance such as a polysaccharide contained in the stool sample is brought in.

Devised to reduce these effects in the pretreatment agent and the PCR reaction solution (Patent Document 1). PCR inhibition by stool has been reported to be improved by the addition of betaine, BSA, T4 gene 32 protein, and protease inhibitor, but RNA spiked in a stool sample was detected and RNA RT- It was unclear whether detection by PCR could be improved (Non-Patent Document 5). The present inventors have already made all or part of a quaternary ammonium salt (betaine, L-carnitine, etc.), bovine serum albumin, glycerol, glycol, and gelatin having a structure in which three methyl groups in amino acids have already been added. It has been found that PCR inhibition by stool can be eliminated by combining.

On the other hand, since heat treatment of the stool sample inactivates reverse transcriptase, it is necessary to add an RT-PCR reaction solution after the heat treatment. Here, the opening and closing of the reaction vessel and the addition of reagents require much labor and labor. In addition, there is a risk of contamination between samples. K. Kang et al. Have reported that highly pathogenic North American porcine genital respiratory syndrome virus RNA can be detected directly from porcine serum samples by RT-PCR (Non-patent Document 6). In this report, the viral envelope structure is destroyed and viral RNA is detected simply by mixing the sample directly with the RT-PCR reaction solution and reacting. However, viruses that do not have an envelope that can infect the digestive tract, such as Norovirus, have RNA retained in a rigid capsid structure that is resistant to inactivation by gastric acid, bile acid surfactant, and the like. Therefore, there are cases where the capsid structure is retained only by subjecting an untreated sample directly to RT-PCR, and RNA cannot be sufficiently detected. Non-Patent Document 7 reports that the destabilizing effect of the capsid structure by the polar organic solvent on the virus differs depending on the type of virus, which makes RNA detection more difficult.

Therefore, there is a method in which the presence or absence of viral RNA can be efficiently detected by adding a chaotropic agent to the RT-PCR reaction solution, without prior RNA isolation or heat treatment from a stool sample or a concentrated sample of a wiping test. However, it is known that the addition of an excessive chaotropic agent inhibits the PCR reaction, and it has been difficult to shorten the PCR reaction time. In addition, with the shortening of the PCR reaction time, there are cases in which the detection sensitivity of norovirus decreases even in the conventional heat treatment method. Therefore, there is a need for a pretreatment method for a specimen containing a viral RNA that is more efficient and hardly inhibits the PCR reaction.

JP2015-119656A

The object of the present invention is to detect the presence or absence of viral RNA with a precision higher than that of a conventionally known method by one-step RT-PCR without isolating non-enveloped virus RNA from the sample or performing preliminary heat treatment of the sample. It is possible to detect this.

As a result of intensive studies in view of the above circumstances, the present inventors have found that non-purified viral RNA or a sample that has not been subjected to prior heat treatment is mixed with a polar organic solvent and then subjected to one-step RT-PCR. It has been found that detection of enveloped virus RNA is possible.
Furthermore, the present inventors determined the polar organic solvent at a concentration necessary for the denaturation of the virus capsid structure and the polar organic solvent concentration allowed for the one-step RT-PCR reaction. After adding RT-PCR reaction solution in order and sealing the reaction vessel, it was found that viral RNA can be detected only by carrying out the reaction in the temperature cycle for RT-PCR, and the present invention has been completed.

The representative invention of the present application is as follows.
Item 1. A method for examining an RNA virus having no envelope in a sample, comprising the following steps without RNA purification from the sample or prior heat treatment of the sample: Virus inspection method for inspection.
(1) A step of mixing a sample and a reagent containing a polar organic solvent,
(2) adding a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase or a DNA polymerase having reverse transcription activity to the mixed solution;
(3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
Item 2. The virus inspection method according to claim 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 after the reaction vessel is sealed in step (3), the one-step RT-PCR reaction is performed without opening and closing the lid.
Item 4. Item 4. The virus inspection method 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 a 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 a wiping test sample in the environment in water, physiological saline, or a buffer solution, and concentrating the suspension.
Item 8. Item 8. The virus inspection method 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 G1 type or 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 virus inspection method 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 variants thereof.
Item 12. Item 1 to 11, wherein the reverse transcriptase is selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The inspection method of the virus in any one.
Item 13. A one-step RT-PCR reaction solution in the step (3) is 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 virus inspection method 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 of 5 mg / ml or more.
Item 14. Item 14. The virus inspection method according to Item 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
Item 15. The virus inspection 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 test kit for a virus having no 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. The item in which the one-step RT-PCR reaction solution in step (3) contains at least one selected from the group consisting of betaine-like quaternary ammonium salts, 0.5 mg / ml or more of bovine serum albumin, glycerol, glycol and gelatin. 16. A kit for testing a virus according to 16.
Item 18. Item 18. The kit for virus inspection according to Item 16 or 17, further comprising a primer pair corresponding to the detection region of the RNA virus to be detected.
Item 19. Item 19. The kit for virus inspection according to any one of Items 16 to 18, further comprising a hybridization probe corresponding to a detection region of the RNA virus to be detected.
Item 20. Item 20. The kit for testing for viruses according to any one of Items 16 to 19, wherein the RNA virus having no envelope is Norovirus.
Item 21. Item 20. The kit for virus inspection according to Item 20, wherein the determination is made as to whether the Norovirus is G1 type or G2 type.

According to the present invention, isolation of viral RNA from a sample and prior heat treatment of the sample are 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 becomes possible to detect the presence or absence of a non-enveloped virus. As a result, since the examination work becomes more efficient, it is possible to increase the examination amount of the subject who has no symptoms even if it is infected with a virus, which contributes to the prevention of infectious diseases. Further, the opening / closing operation of the reaction vessel lid is also omitted due to the omission of the heat treatment step. As a result, the risk of scattering of the virus-containing sample when the lid is opened and closed can be eliminated, and the risk of contamination with other samples can also be reduced. As a result, the risk of false positives can be suppressed, and the accuracy of 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. It is a figure which shows the result of having examined the specimen pretreatment effect 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 operation | work 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-envelope RNA virus such as Norovirus in a sample, in which a sample and a reagent containing a polar organic solvent are mixed without performing purification of the viral RNA from the sample or prior heat treatment of the sample. And a method for examining the presence or absence of a virus, comprising adding a one-step RT-PCR reagent containing reverse transcriptase and DNA polymerase, or DNA polymerase having 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, characterized in that it comprises at least the following steps.
(1) A step of mixing a sample and a reagent containing a polar organic solvent,
(2) adding a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase or a DNA polymerase having reverse transcription activity to the mixed solution;
(3) A step of carrying out 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 the steps (1) and (2). The total amount of the mixed solution may be subjected to the step (2) by the step (1), or a part thereof may be transferred to another container and the step (2) may be performed. Furthermore, in step (3), it is preferable not to open and close the lid of the reaction vessel after sealing the reaction vessel.

The RNA virus to be examined in the present invention is not particularly limited as long as it is a non-enveloped RNA virus that does not have an envelope derived from a lipid bilayer. Such non-envelope RNA viruses include Astroviridae Astrovirus, Caliciviridae Sapovirus, Norovirus, Picornaviridae Hepatitis A virus, Echovirus, Enterovirus, Coxsackie virus, Poliovirus, Rhinovirus, Hepe Examples include Viridae hepatitis E virus, Reoviridae rotavirus, and the like, but are not particularly limited, and are particularly useful for detecting norovirus. Most non-enveloped viruses can infect the gastrointestinal tract by fecal infection, etc., and RNA is retained in a rigid capsid structure that is resistant to inactivation by gastric acid and bile acid surface-active action. For this reason, a non-enveloped virus may not be inactivated even under conditions where an enveloped virus (such as influenza virus) is inactivated.

Examples of the sample used in the present invention include feces (excretion feces, rectal stool), vomit, saliva and the like, but are not particularly limited, and can be used for all samples derived from living bodies. In particular, it is useful for detection from feces (excretion feces, rectal feces). Feces are characterized by containing a large amount of proteins and nucleic acids derived from E. coli in addition to proteases and nucleolytic enzymes as contaminants. It is known that reaction solution components such as enzymes, primers, and nucleic acid probes used in RT-PCR reactions are digested or inactivated due to the influence of contaminants contained in feces, and the detection sensitivity is lowered. In the present invention, it is not necessary to isolate RNA from these samples with a commercially available RNA purification kit, or to perform heat treatment in advance to expose RNA from the virus structure by heat treatment. The sample may be directly used for detection, or it may be a sample in which the sample is suspended in water, physiological saline or a buffer solution in order to reduce the influence on the reaction of contaminants and obtain a more stable test result. There may be. Furthermore, in the case of a sample having a large amount of impurities such as stool, the supernatant may be used after centrifugation. Alternatively, filter filtration may be performed. The buffer solution is not particularly limited, and examples include Hanks buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and tricine buffer solution.

A sample according to another aspect of the present invention is a wipe inspection sample. Wiping inspection is useful for elucidating the pollution route and grasping the contamination status of the facility environment. In the present invention, the wiping inspection is not particularly limited, but is a sample that is wiped off the corresponding compartment or equipment with a cotton swab, etc., eluted in water or a buffer solution, and concentrated with polyethylene glycol (PEG) precipitation or the like. . Specific examples of the wiping inspection procedure include "Improvement of norovirus inspection method for wiping specimens" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). There is no particular limitation, and a method equivalent to this is widely included. Examples of wiping points include cooking utensils such as chopping boards, kitchen knives, dishwashers, tableware, refrigerator handles and toilets, bathroom doorknobs, toilets, kitchens, toilets, bathroom faucets, cook's hands and fingers, bathroom , Facilities such as toilets, washbasins, handrails, and living rooms. Moreover, although it is not a wiping test | inspection, it can apply also to the concentrated sample of a sewage sample as an environmental test | inspection.

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 a negative charge in the molecule does not coincide is called a polar molecule. A solvent composed of polar molecules is called a polar solvent. Among polar solvents, the use of a polar organic solvent composed of an organic compound can destabilize higher-order structures of biomolecules such as nucleic acids and proteins. By utilizing this property, the capsid structure is destabilized by weakening the hydrophobic bond of the capsid protein of the virus. As described above, it is known that the destabilizing effect of the capsid structure by the polar organic solvent on the virus varies depending on the type of virus (Non-patent Document 7). This is presumably because the strength of the hydrophobic bond varies depending on the difference in the properties of the capsid protein possessed by the virus.

Specific examples of the polar organic solvent include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethyl sulfoxide, acetone, acetonitrile, ethanol , Methanol, 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Preferred are methanol, triethylamine, dimethyl sulfoxide, and acetone. Further, it may be a mixed solution containing two or more of these polar organic solvents. The lower limit concentration of the polar organic solvent as a capsid protein denaturant is not particularly limited as long as it is a concentration at which the capsid protein is denatured, although it depends on the type of polar organic solvent and other additives. In addition, since the capsid protein varies depending on the type of virus, the effective concentration of the polar solvent varies from virus to virus. Usually, the effective concentration of the polar solvent relative to the amount of the sample is 10% or more and less than 100%, more preferably 30. % 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,

The polar organic solvent is usually known as a PCR inhibitor. Therefore, the polar organic solvent, sample, and 1-step RT-PCR reaction solution are added in sequence by selecting the polar organic solvent that has a small difference between the concentration necessary for protein denaturation and the allowable concentration for PCR. Thus, 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. As an example of such a polar organic solvent, dimethyl sulfoxide is particularly preferable. For example, when 1 μL of dimethyl sulfoxide and 1 μL of a sample are mixed and 48 μL of 1-step RT-PCR solution is added, the concentration of dimethyl sulfoxide brought into the reaction solution is 2%. 2% dimethyl sulfoxide is an acceptable concentration even when RT-PCR solution is brought in.

* Opening and closing of the reaction vessel occurs during sample transfer or heat treatment. The opening and closing operation of the reaction vessel is complicated and increases the working time. In addition, when the reaction vessel containing the virus-containing specimen is opened and closed, there is a risk of scattering of the virus and virus-derived RNA. The scattering of virus not only threatens the safety and health of workers, but also means contamination of the inspection work environment. Since the scattered RNA virus is aerosolized in the workplace, the risk of contamination of other samples being examined simultaneously is a problem. For this reason, the method for examining the presence or absence of viruses using RT-PCR without a lid opening / closing step has significance more than simplification of work.

The 1-step RT-PCR solution added to the mixed solution contains reverse transcriptase and DNA polymerase. It is preferable to use Tth DNA polymerase or Taq DNA polymerase, which are DNA polymerases having reverse transcriptase activity. More preferably, two enzymes are used, and at least two enzymes, reverse transcriptase and 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 into DNA. However, MMLV (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV -RT, RAV2-RT, EIAV-RT, Carboxyothermus hydrogenformaman 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. There is no particular limitation. More preferably, Taq, Tth or a variant 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 reaction, the enzyme activity of DNA polymerase during reverse transcription reaction by introducing into heat-labile blocking group DNA polymerase together with anti-DNA polymerase antibody or by chemical modification It is preferable that application to hot start PCR is possible.

The 1-step RT-PCR reaction solution used in the present invention includes a reverse transcriptase and a DNA polymerase, as well as a buffer, an appropriate salt, magnesium salt or manganese salt, deoxynucleotide triphosphate, and viral RNA to be detected. A primer pair corresponding to the detection target region is included, and an additive may be further included as necessary.

The buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, and Bicine. The pH is adjusted to 6 to 9, more preferably pH 7 to 8, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid or the like. The concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added in order to obtain 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 dNTPs 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 as an alternative and / or part of dTTP. Examples of magnesium salts include magnesium chloride, magnesium sulfate, magnesium acetate, and manganese salts. Manganese chloride, manganese sulfate, manganese acetate, and the like are exemplified, and about 1 to 10 mM is preferably added.

Furthermore, 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 is preferable to contain at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin of 0.5 mg / ml or more.

Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine), L-carnitine, and the like. If the quaternary ammonium salt has a structure in which three methyl groups are added to the amino group of an amino acid, It is not particularly limited. The structure of betaine-like quaternary ammonium salt is a compound having both positive and negative charges which are stable in the molecule, and exhibits a property like a surfactant, which is considered to cause instability of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase. The concentration of the betaine-like quaternary ammonium salt is preferably 0.1M to 2M, more preferably 0.2M to 1.2M.

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

The gelatin contained in the one-step RT-PCR reaction solution is considered to contribute to the stabilization of the PCR enzyme from the skin, bones, tendons, or fish scales and skins of animals such as cows and pigs. The concentration used is preferably such that it does not interfere with fluorescence detection while stabilizing PCR amplification. Preferably it is 1 to 5%, more preferably 1 to 2%. The origin of gelatin is not particularly limited, but a fish-derived one is preferable to a cow or pork-derived one because it has a low jelly strength and a good handling of the reaction solution.

Furthermore, it can also be used in combination with a substance known to promote RT-PCR in the technical field. 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 acetic acid 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. Further, 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 primer pair corresponding to the detection region of the RNA virus to be detected, and two pairs of primers in which one primer is complementary to the DNA extension product of the other primer. Can be mentioned. Moreover, what is called multiplex PCR in which 2 or more pairs of the said primer are contained as another aspect is also mentioned. Furthermore, when the target nucleic acid is of a subtype, a degenerate primer may be included. In the present invention, when detecting a norovirus which is one of RNA viruses having no envelope, as an example of a primer pair, as a primer for detecting norovirus, a notification from the Ministry of Health, Labor and Welfare Although the primer as described in the supervisor 1105001) is mentioned, it is not restricted to this.

In another aspect, the present invention is a detection method further comprising at least one type of labeled hybridization probe or a double-stranded DNA-binding fluorescent compound. As a result, the analysis of the amplification product can be monitored not by normal electrophoresis but by monitoring the fluorescence signal, and the analysis effort is reduced. Furthermore, it is not necessary to open the reaction vessel, and the risk of contamination can be reduced. It is also possible to distinguish viral subtypes by labeling each hybridization probe corresponding to a viral subtype with a different fluorescent dye.

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), EvaGreen (registered trademark; Biotium). LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science), and the like.

Examples of hybridization probes used in the present invention include TaqMan hydrolysis probes (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 pamphlet, WO 97/46712 pamphlet). , International Publication No. 97/46714 pamphlet). Examples of the base sequence of the probe for detecting norovirus include, but are not limited to, the sequence described in the notice (food safety supervisor 1105001) of the Ministry of Health, Labor and Welfare, Pharmaceutical Food Bureau, Safety Department, Monitoring and Safety Section. Furthermore, when the target nucleic acid consists of subtypes, 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 A test kit for RNA viruses having no envelope, characterized in that The kit for testing a virus of the present invention comprises at least 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 preferably contains at least one of betaine-like quaternary ammonium salt, 0.5 mg / ml or more of 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 further 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.

Example 1. Screening of Samples Required for Heat Treatment (1) RT-PCR Reaction Solution The reaction solution having the composition shown below was used as a basic composition, and the influence of organic solvents was evaluated.
Reaction solution 30μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Primer solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Probe solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Pretreatment solution 4μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)

(2) Sample Preparation Method 15 specimens of human stool positive for Norovirus G2 were suspended in 0.5 ml of sterilized water so as to be 10% (weight%). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.

(3) Reaction Under conditions for carrying out 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 heat treatment at 85 ° C. for 1 minute using a thermal cycler (Gene Amp (registered trademark) PCR System 9700) manufactured by Applied Bioscience. 45 μL of the RT-PCR reaction solution (reaction solution + primer solution + probe solution + enzyme solution) was added to the pretreated solution after the heat treatment to obtain a PCR reaction solution. Under conditions where no pretreatment was performed, 4 μL of the pretreatment solution was added to 45 μL of the RT-PCR reaction solution, and 1 μL of each of the prepared samples was added to obtain a PCR reaction solution without any heat treatment. Using this, CFX96WELL DEEP manufactured by BioRad was used to carry out a real-time PCR reaction in the following temperature cycle. Fluorescence readings were taken in the last 30 cycles of a 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 22 seconds 10 cycles (PCR)
95 ° C 1 second-54 ° C 22 seconds 30 cycles (PCR)

(4) Results The probe solution (Norovirus detection kit G1 / G2-high-speed probe detection—attached to (Toyobo)) detects the internal control gene in the FAM channel, Norovirus G1 in the Cy5 channel, and Norovirus G2 in the ROX channel. Here, Cq values of G1 and G2 RNA are shown. As a result, it was confirmed that all G2-positive specimens were detected under the heat treatment conditions (Table 1). On the other hand, in the samples that were not subjected to the pretreatment, detection could not be confirmed in 11 samples (Table 2). This is probably because the heat treatment was not performed, the virus capsid structure in the specimen was not broken, and RNA molecules did not come out of the virus particles. In this examination, a sample that could not confirm a G2-positive signal was determined as a sample requiring heat treatment.

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

(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 Benzene

(3) Reaction 2 μL of the solvent was added to each reaction tube, 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 synthetic RNA of Norovirus G2. Using this, CFX96WELL DEEP manufactured by BioRad was used to carry out a real-time PCR reaction in the following temperature cycle. Fluorescence readings were taken in the last 30 cycles of a 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 22 seconds 10 cycles (PCR)
95 ° C 1 second-54 ° C 22 seconds 30 cycles (PCR)

(4) Results FIG. 1 shows the Cq value of the resulting G2 RNA. 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, it was confirmed that anisole significantly increased the Cq value compared to the distilled water addition condition, and PCR inhibition occurred.

Example 3 Screening of solvent having pretreatment effect on virus-containing specimen (1) RT-PCR reaction solution The reaction solution having the composition shown below was used as a basic composition, and the pretreatment effect of the specimen of each solvent was evaluated.
Reaction solution 30μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Primer solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Probe solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Pretreatment solution 4μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)

(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 Acetyl-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 solvent 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 this, CFX96WELL DEEP manufactured by BioRad was used to carry out a real-time PCR reaction in the following temperature cycle. Fluorescence readings were taken in the last 30 cycles of a 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 22 seconds 10 cycles (PCR)
95 ° C 1 second-54 ° C 22 seconds 30 cycles (PCR)

(5) Results FIG. 2 shows the Cq value of the resulting G2 RNA. As a result, no detection was confirmed with ethanol, 3-methylbutyl acetate, anisole, hexane, and benzine, and no pretreatment effect on the virus-containing specimen was confirmed. In contrast, detection of G2 signal was confirmed with methanol, acetone, triethylamine, and dimethyl sulfoxide. In particular, dimethyl sulfoxide was confirmed to have a high pretreatment effect on a virus-containing specimen because the Cq value was 3 or more lower than urea having a pretreatment effect.

Example 4 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 as a pretreatment effect on a virus-containing specimen was evaluated.
Reaction solution 30μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Primer solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Probe solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Pretreatment solution 4μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)

(2) Sample Preparation Method Norovirus G2-positive human stool specimens e1, f1, and h2, which are specimens required for 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 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. Mixed with.
Condition 1 1 μL of each sample, 2 μL of 100% dimethyl sulfoxide (effective concentration 67%)
Condition 2 Each sample 1 μL, 90% dimethyl sulfoxide 2 μL (effective concentration 60%)
Condition 3 1 μL of each sample, 2 μL of 80% dimethyl sulfoxide (effective concentration 53%)
Condition 4 Each sample 1 μL, 70% dimethyl sulfoxide 2 μL (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 Each sample 1 μL, 40% dimethyl sulfoxide 2 μL (effective concentration 27%)
Condition 8 1 μL of each sample, 2 μL of distilled water

(4) Reaction It added that 2 microliters of each dimethyl sulfoxide aqueous solution of the said conditions was added to the reaction tube, and it was confirmed that it fell 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 this, CFX96WELL DEEP manufactured by BioRad was used to carry out a real-time PCR reaction in the following temperature cycle. Fluorescence readings were taken in the last 30 cycles of a 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 22 seconds 10 cycles (PCR)
95 ° C 1 second-54 ° C 22 seconds 30 cycles (PCR)

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

Example 5 FIG. Study on 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 influence of the amount of dimethyl sulfoxide carried on RT-PCR was evaluated.
Reaction solution 30μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Primer solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Probe solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Enzyme solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)

(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 To the reaction tube, dimethyl sulfoxide and distilled water were added in the volumes 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 RNA of Norovirus G2 and G1 were added. Using this, CFX96WELL DEEP manufactured by BioRad was used to carry out a real-time PCR reaction in the following temperature cycle. Fluorescence readings were taken in the last 30 cycles of a 54 ° C. extension step.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 10 seconds,
95 ° C 1 second-54 ° C 22 seconds 10 cycles (PCR)
95 ° C 1 second-54 ° C 22 seconds 30 cycles (PCR)

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

Example 6 Comparison of dimethyl sulfoxide treatment and conventional pretreatment method (1) RT-PCR reaction solution Pretreatment of virus-containing specimens with dimethyl sulfoxide under the conditions where the reaction composition of the following composition is the basic composition and the PCR reaction time is shortened The effect was compared with the conventional pretreatment method.
Reaction solution 30μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Primer solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Probe solution 5μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)
Enzyme solution 5μL
0.8 unit / μL rTth mutant DNA polymerase
0.24 μg / μL Anti-Tth antibody (Toyobo)
1 unit / μl ReverseAce (Toyobo)
Pretreatment solution 4μL
(Norovirus detection kit G1 / G2-High-speed probe detection-(Toyobo) attachment)

(2) Sample Preparation Method Norovirus G2-positive human stool specimens e2 and g2, which are specimens required for 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 treatment with dimethyl sulfoxide After mixing 2 μL of dimethyl sulfoxide and 1 μL of the sample, 45 μL of the 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 fluorescence value was read in the 54 ° C. extension step of the latter half 30 cycles.

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

(5) Detection by heat treatment method 4 μL of the pretreatment liquid 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), using CFX96WELL DEEP manufactured by BioRad, real-time PCR reaction was performed at the temperature cycle described in (6). Fluorescence readings were taken in a 30 cycle 54 ° C extension step.

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

(7) Results FIG. 3 shows the Cq value of the G2 signal when each pretreatment method is carried out. In this study, the pretreatment method for obtaining the G2 signal was only treatment with dimethyl sulfoxide. As the RT-PCR reaction time is shortened, the efficiency of disruption of the capsid structure of the virus is low, and the amount of RNA used as a template is insufficient under the condition that the leakage of RNA into the reaction solution is small. The reason is that it did not respond sufficiently. Therefore, compared with the conventionally known heat treatment method and chaotropic agent treatment method, the dimethyl sulfoxide treatment method of the present invention showed that the capsid crushing efficiency of the virus in the specimen was high.

Example 7 Comparison of working time of dimethyl sulfoxide treatment and conventional heat treatment method (1) Work process The main work steps of the pretreatment method including dimethyl sulfoxide treatment and heat treatment step are shown in FIG. A special jig (Fast Gene Cap easy) manufactured by Nippon Genetics Co., Ltd. was used to open and close 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 5 times, and the average value was calculated and compared.

(3) Results In the conventional method including the heat treatment step, it takes about 21 minutes and 30 seconds, whereas in the dimethyl sulfoxide treatment of the present invention, it takes about 14 minutes, resulting in a working time reduction of 36%. (FIG. 5).
The RT-PCR reaction time is 45 minutes when the following reaction cycle is carried out 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 1 second-54 ° C 5 seconds 30 cycles (PCR)
When combined with the time required for the pretreatment method using dimethyl sulfoxide treatment, it takes 59 minutes, and the time required from the sample treatment to the measurement can be achieved within 1 hour.

The present invention is suitably used in molecular biology research, and further in examinations for clinical examination and food hygiene management.

Claims (21)

1. A method for examining an RNA virus having no envelope in a sample, comprising the following steps without RNA purification from the sample or prior heat treatment of the sample: Virus inspection method for inspection.
   (1) A step of mixing a sample and a reagent containing a polar organic solvent,
   (2) adding a one-step RT-PCR reaction solution containing reverse transcriptase and DNA polymerase or a DNA polymerase having reverse transcription activity to the mixed solution;
   (3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
2. The virus inspection method according to claim 1, wherein the steps (1) and (2) are performed in the same container.
3. The virus inspection method according to claim 1 or 2, wherein after the reaction vessel is sealed in step (3), the one-step RT-PCR reaction is performed without opening and closing the lid.
4. The virus inspection method according to any one of claims 1 to 3, wherein the sample is feces.
5. The virus inspection method according to any one of claims 1 to 3, wherein the sample is a suspension suspended in water, physiological saline or a buffer solution.
6. The virus inspection method according to any one of claims 1 to 3, wherein the sample is a centrifugal supernatant of a suspension.
7. The sample of the virus according to any one of claims 1 to 3, wherein the sample is a sample obtained by suspending a wiping test sample in the environment in water, physiological saline or a buffer solution, and concentrating the suspension. Inspection method.
8. The virus inspection method according to any one of claims 1 to 7, wherein the RNA virus having no envelope is a norovirus.
9. The virus inspection method according to claim 8, wherein it is determined whether the Norovirus is G1 type or G2 type.
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. The virus inspection method according to claim 1, wherein the virus inspection method is any one of the above.
11. The method for examining 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 variants thereof.
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 inspection method of the virus in any one of.
13. A one-step RT-PCR reaction solution in the step (3) is 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 of 5 mg / ml or more.
14. The virus inspection method according to claim 13, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
15. The virus inspection 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.
16. A test kit for a virus having no envelope, comprising a reagent containing a polar organic solvent, a reverse transcriptase, a DNA polymerase, and a one-step RT-PCR reaction solution.
17. The one-step RT-PCR reaction solution in step (3) comprises at least one selected from the group consisting of betaine-like quaternary ammonium salts, 0.5 mg / ml or more of bovine serum albumin, glycerol, glycol and gelatin. Item 17. A kit for testing a virus according to Item 16.
18. The virus test kit according to claim 16 or 17, further comprising a primer pair corresponding to a detection region of the RNA virus to be detected.
19. The virus test kit according to any one of claims 16 to 18, further comprising a hybridization probe corresponding to a detection region of the RNA virus to be detected.
20. 20. The virus testing kit according to claim 16, wherein the RNA virus having no envelope is a norovirus.
21. 21. The test virus kit according to claim 20, wherein it is determined whether the Norovirus is G1 type or G2 type.

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