JP2017209036A - Improved virus detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a kit, which enable the detection of the presence or absence of viral RNA by one step RT-PCR in addition to a reaction solution without isolation of non-enveloped virus RNA from a sample or preliminary heat treatment of the sample.SOLUTION: According to the present invention, there is provided a method for testing RNA virus having no envelope in a sample, the method examines the presence or absence of virus without isolation of non-enveloped virus RNA from a sample or preliminary heat treatment of the sample. The method comprises the steps of: (1) mixing a sample and a reagent containing a chaotropic agent; (2) adding a RT-PCR reaction solution containing a reverse transcriptase and a thermostable DNA polymerase to the mixed solution; and (3) performing a one-step RT-PCR reaction after sealing a reaction vessel.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting a virus by nucleic acid amplification. More specifically, a fecal sample or a sample obtained by adding a reaction solution of real-time reverse transcription polymerase chain reaction (qRT-PCR) after mixing the sample with a chaotropic agent without isolating nucleic acid from the sample or heat treatment. It relates to the detection of viruses without envelopes in environmental wipe samples. 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 RNA is analyzed, 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. -PCR, (3) The enzyme is roughly divided into three types, ie, a two-enzyme system, one-step RT-PCR, in which RT and PCR are successively performed using two types of enzymes, reverse transcriptase and DNA polymerase.

  Among RT-PCR, in the genetic test, one-step RT-PCR is preferred in order to avoid high contamination and contamination due to opening and closing of the reaction container during the reaction. In the one-enzyme system one-step RT-PCR, a DNA polymerase having 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, reverse transcriptase derived from retrovirus or bacteria (for example, MMLV (Moloney Murine Leukemia Virus) -RT (Reverse Transscriptase), AMV (Avian Myeloblastosis virus) avian myeloblast virus virus; -RT, HIV (Human Immunity Viral) -RT, RAV (Rouse-associated virus) 2-RT, EIAV (Equine Infectious Anneavirus) -RT, Carboxidothermus hydrogenoform (C) These variants are utilized. As a DNA polymerase. Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof are used.

One-enzyme one-step RT-PCR method is known to be less sensitive than 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 derived from retrovirus has higher reverse transcription efficiency 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 the 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.

  Norovirus, 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 these, the RT-PCR method based on the notification (safety supervisor 1105001) of the monitoring and safety section of the Ministry of Health, Labor and Welfare, Pharmaceutical Food Bureau, Safety Department 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 having 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. Therefore, a device for reducing these influences has been applied to 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 inventor has already made a quaternary ammonium salt (betaine, L-carnitine, etc.) having a structure in which three methyl groups are added in amino acids, 1 mg / mg or more of bovine serum albumin, glycerol, glycol, and gelatin. It has been found that PCR inhibition by stool can be eliminated by combining all or part of it.

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, 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 the 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. Therefore, there has been a demand for a method for efficiently detecting the presence or absence of viral RNA in addition 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.

JP2015-119656A

BioTechniques, Vol. 17, 1994, pp. 1034-1036 BioTechniques, 25, 1998, 230-234 Nucl. Acids Res. 20, 1992, pp. 1487-1490. J. et al. Virol. Methods, 163, 2010, 282-286. J. et al. Virol. Microbiol. 38, 2000, 4463-4470. J. et al. Animal Science And Biotechnology, Vol. 5, 2014, p. 45

The object of the present invention is to enable detection of the presence or absence of viral RNA by one-step RT-PCR in addition to the reaction solution, without isolating non-enveloped viral RNA from the sample or prior heat treatment of the sample. It is.

As a result of intensive studies in view of the above circumstances, the present inventors have mixed non-enveloped samples with a chaotropic agent after being subjected to purification of virus RNA and prior heat treatment, thereby providing a non-envelope. It was found that detection of viral RNA is possible.
Furthermore, the present inventors determined the concentration of the chaotropic agent necessary for the denaturation of the virus capsid structure and the concentration of the chaotropic agent allowed for the one-step RT-PCR reaction, and placed the chaotropic agent, the sample, and the one-step RT-PCR in the same reaction vessel. It was found that viral RNA can be detected simply by carrying out the reaction in a temperature cycle for RT-PCR after sequentially adding the reaction solution and sealing the reaction vessel, thereby completing the present invention.

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, the method comprising examining the presence or absence of a virus comprising the following steps without RNA purification from the sample or prior heat treatment of the sample.
(1) A step of mixing a sample and a reagent containing a chaotropic agent,
(2) adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a thermostable DNA polymerase to the mixed solution;
(3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
Item 2. Item 2. The method according to Item 1, wherein the steps (1) and (2) are performed in the same container.
Item 3. Item 3. The method according to any one of Items 1 and 2, wherein the sample is feces.
Item 4. Item 4. The 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 5. The method according to claim 1, wherein the sample is a centrifugal supernatant of the suspension according to claim 4.
Item 6. Item 3. The method according to Item 1 or 2, wherein the sample is a sample obtained by suspending a wiping test sample in the environment in water, physiological saline or buffer, and concentrating the suspension.
Item 7. Item 7. The method according to any one of Items 1 to 6, wherein the virus having no envelope is Norovirus.
Item 8. Item 8. The method according to Item 7, wherein it is determined whether the Norovirus is G1 type or G2 type.
Item 9. Item 9. The method according to any one of Items 1 to 8, wherein the chaotropic agent is at least one selected from the group consisting of urea, thiourea, guanidine thiocyanate, guanidine hydrochloride, and arginine.
Item 10. Item 10. The method according to any one of Items 1 to 9, wherein the thermostable DNA polymerase is selected from Taq, Tth or a variant thereof.
Item 11. Item 11. The method according to any one of Items 1 to 10, wherein the origin of reverse transcriptase is selected from Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV), or a variant thereof.
Item 12.1 Step-RT-PCR reaction solution is a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as “betaine-like quaternary ammonium”), 1 mg / ml or more Item 12. The method according to any one of Items 1 to 11, comprising at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin.
Item 13. Item 13. The method according to Item 12, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
Item 14. A test kit for a virus having no envelope, comprising a reagent containing a chaotropic agent, a reverse transcriptase, a thermostable polymerase, and a one-step RT-PCR reaction solution.
Item 15. The kit according to Item 14, wherein the step-RT-PCR reaction solution comprises at least one selected from the group consisting of betaine-like quaternary ammonium salts, 1 mg / ml or more of bovine serum albumin, glycerol, glycol and gelatin. .
Item 16. Item 16. The kit according to Item 14 or 15, further comprising a primer pair corresponding to the detection region of the virus to be detected.
Item 17. The kit according to any one of claims 14 to 16, further comprising a hybridization probe corresponding to a detection region of a virus to be detected.
Item 18. Item 18. The kit according to any one of Items 14 to 17, wherein the virus having no envelope is a norovirus.
Item 19. Item 21. The kit according to Item 20, wherein it is determined whether the norovirus is G1 type or G2 type.

According to the present invention, isolation of viral RNA from a sample or prior heat treatment of the sample is not required, and the sample is mixed with a chaotropic agent and then added to a one-step RT-PCR reaction solution, such as norovirus in the sample. It becomes possible to detect the presence or absence of non-enveloped viruses. 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.

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 in a sample such as Norovirus, in which a sample and a reagent containing a chaotropic agent are mixed without purifying the viral RNA from the sample or prior to heat treatment of the sample. This is a method for examining the presence or absence of a virus comprising adding a one-step RT-PCR reagent containing reverse transcriptase and heat-resistant DNA polymerase.

The method for examining the presence or absence of the virus of the present invention is a method for examining the presence or absence of a virus comprising the following steps.
(1) A step of mixing a sample and a reagent containing a chaotropic agent,
(2) adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a thermostable DNA polymerase 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. Between the steps (1) and (2), it is preferable not to transfer all or part of the mixed solution to another container. That is, the total amount of the mixed solution may be provided 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.

  The RNA virus to be tested in the present invention is a non-enveloped RNA virus that does not have an envelope derived from a lipid bilayer membrane. 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. 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.

  Examples of the sample of the present invention include stool (excretion stool, rectal stool), vomit, saliva and the like, but are not particularly limited. These samples in the present invention are characterized in that it is not necessary to isolate RNA 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 with many 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.

  Another embodiment 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, a chaotropic agent refers to a substance having a property of destabilizing a molecular structure by affecting hydrogen bonds, van der Waals forces, hydrophobic bonds, and the like. By using a chaotropic agent, higher-order structures of biomolecules such as nucleic acids and proteins can be destabilized. By utilizing this property, the capsid structure is destabilized by weakening the hydrophobic bond of the capsid protein of the virus.

Specific examples of the chaotropic agent include guanidine thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, urea, thiourea, arginine, sodium iodide and the like, but are not limited thereto. The lower limit concentration of the chaotropic agent 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 chaotropic agent and other additives. .3M to 8M, more preferably 0.5M to 8M, and still more preferably 1M to 8M.

  The chaotropic agent may be combined with one or more surfactants, reducing agents, chelating agents,

  The chaotropic agent is also known as a PCR inhibitor. Therefore, among the chaotropic agents, the one that has a small difference between the concentration required for protein denaturation and the permissible concentration for PCR is selected, and the chaotropic agent, the sample, and the one-step RT-PCR reaction solution are sequentially added. Thus, the detection operation can be easily carried out in the same container from the denaturation of the capsid protein to the one-step RT-PCR reaction without opening and closing the container. An example of such a chaotropic agent is urea. For example, when 2.5 μl of 8M urea and 1 μl of a sample are mixed and 46.5 uL of 1-step RT-PCR solution is added, urea brought into the reaction solution is 0.4M. 0.4M urea is an acceptable concentration even when RT-PCR solution is brought in. On the other hand, when 1M guanidine thiocyanate is used in the same amount of operation liquid, 50 mM guanidine thiocyanate is brought into the one-step RT-PCR reaction. As a result, since the RT-PCR reaction is inhibited, it is necessary to partially transfer the mixed solution of guanidine thiocyanate and the sample, and the operability is lowered.

  The one-step RT-PCR solution added to the mixed solution contains reverse transcriptase and thermostable DNA polymerase. A thermostable DNA polymerase having a reverse transcriptase activity, Tth DNA polymerase, Taq DNA polymerase, or the like may be used, but more preferably, two enzymes, at least two enzymes, reverse transcriptase and DNA polymerase are used. is there.

  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, but MMLV (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV Examples include -RT, RAV2-RT, EIAV-RT, Carboxyothermus hydrogenoforman DNA polymerase) and mutants thereof. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.

Examples of the thermostable 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. However, it is not particularly limited. 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.

In addition to reverse transcriptase and thermostable DNA polymerase, buffer solution, appropriate salt, magnesium salt or manganese salt, deoxynucleotide triphosphate, virus RNA to be detected The primer pair corresponding to the detection target region may further contain an additive as necessary.

Although it does not specifically limit as a buffering agent used by this invention, Tris (Tris), Tricine (Tricine), Bis-Tricine (Bis-Tricine), Bicine (Bicine), etc. are mentioned. 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. Moreover, as a density | concentration of the buffer agent to add, it is 10-200 mM, More preferably, it is used at 20-150 mM. At this time, a salt solution is added in order to obtain ionic conditions suitable for the reaction. As the salt solution, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like are added.

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 the magnesium salt include magnesium chloride, magnesium sulfate, magnesium acetate, and manganese salt. 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 desirable to contain at least one selected from the group consisting of bovine serum albumin, glycerol, glycol and gelatin at 1 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.

Gelatin contained in the one-step RT-PCR reaction solution is considered to contribute to the stabilization of the PCR enzyme from the skin, bone, tendon, or fish scale and skin 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 substances that promote RT-PCR known in the art. 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, but is not limited thereto. 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.

  Examples of the primer pair used in the present invention include a pair of two primers in which one primer is complementary to the DNA extension product of the other primer. 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 viruses having no envelope, as a primer pair for detecting a norovirus, a notification from the Ministry of Health, Labor and Welfare, the Ministry of Health, Labor and Welfare, Safety Department of the Ministry of Health, Labor and Welfare However, the primer is not limited to this.

Another aspect of 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 aspect of the present invention is a kit for testing viral RNA in a sample, comprising a chaotropic agent, a reverse transcriptase and a thermostable DNA polymerase, and a one-step RT-PCR reaction solution. This is a test kit for viruses without an envelope.

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

Example 1.1 Examination of allowable amount of chaotropic agent to be introduced in step RT-PCR reaction (1) Reaction solution having the following composition as the basic composition, and the influence of guanidine thiocyanate and urea shown in (2) evaluated.
1xBuffer (Blend Taq (registered trademark) attachment buffer (Toyobo))
0.3 M L-carnitine 1% Gelatin 1 mg / ml BSA
Each 0.2 mM dATP, dGTP, dCTP, dTTP
Primer solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
Probe solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
0.05 unit / μl rTth DNA polymerase (Toyobo)
0.01 μg / μl Anti-Tth antibody 0.05 unit / μl ReversetraAce (Toyobo)
(2) Chaotropic agent Condition 1 No chaotropic agent Condition 2 160 mM guanidine thiocyanate Condition 3 80 mM guanidine thiocyanate Condition 4 40 mM guanidine thiocyanate Condition 5 20 mM guanidine thiocyanate Condition 6 10 mM guanidine thiocyanate Condition 7 No chaotropic agent Condition 8 160 mM urea Condition 9 320 mM urea Condition 10 480 mM urea Condition 11 640 mM urea Condition 12 800 mM urea Condition 13 960 mM urea Condition 14 1120 mM urea Condition 15 1280 mM urea (3) reaction 1250 of the synthesized RNA of Norovirus G1 and G2 in 49 μl of the reaction solution Copy, 250 copies, 50 copies (N = 2), 25 copies (N = 2) were added. This was subjected to real-time PCR reaction using BioRad CFX96WELL DEEP at the following temperature cycle. The fluorescence value was read in the extension step of 60 ° C. in the latter half 30 cycles.
42 ° C for 15 minutes (reverse transcription reaction),
95 ° C for 20 seconds,
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 10 cycles (PCR)
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 30 cycles (PCR)
(4) Results In the probe solution (Norovirus detection kit G1 / G2-probe detection— (Toyobo) accessory), the FAM channel detects the internal control gene, the Cy5 channel detects Norovirus G1, and the ROX channel detects Norovirus G2. Here, Tables 1 and 2 show Cq values of G1 and G2 RNA. As a result, guanidine thiocyanate completely inhibits the RT-PCR reaction at 80 mM or more. The allowable amount of guanidine thiocyanate was 40 mM, more preferably about 20 mM. For urea, 25 copies of RNA could not be detected at 960 mM or higher. The allowable amount of urea brought in was 800 mM, more preferably 640 mM, and further preferably 480 mM.

Example 2 Effect of mixing sample with guanidine thiocyanate (1) Preparation method of sample Norovirus G2-positive human stool 4 specimens a8, b5, d6, d7 to 10% (wt%) in 0.5 ml of sterilized water Suspended. This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.
(2) Mixing method with guanidine thiocyanate Condition 1 Samples a8 and b50.5 μL
Condition 2 0.5 μL of 2M guanidine thiocyanate and 0.5 μL of samples a8 and b5
Condition 3 0.4 M guanidine thiocyanate 2.5 μL and samples a8 and b5 0.5 μL
Condition 4 0.285 M guanidine thiocyanate 3.5 μL and samples a8 and b5 0.5 μL
Condition 5: 0.2 μM 0.222M guanidine thiocyanate 4.5 μL and samples a8, b5 0.5 μL
Condition 6: 9.5 μL of 0.105 M guanidine thiocyanate and 0.5 μL of samples a8 and b5
Condition 7 Samples d6 and d7 1 μL
Condition 8 0.625M guanidine thiocyanate 2 μL and samples d6, d7 1 μL
Condition 9 0.5 M guanidine thiocyanate 2 μL and samples d6, d7 1 μL
Condition 10: 0.5 M guanidine thiocyanate 2.5 μL and samples d6 and d7 1 μL
Condition 11 2 M guanidine thiocyanate 0.5 μL and samples d6 and d7 1 μL
(3) Reaction liquid 50 μl of the following reaction liquid was added to the mixed liquid.
1xBuffer (Blend Taq (registered trademark) attachment buffer (Toyobo))
0.3 M L-carnitine 1% Gelatin 1 mg / ml BSA
Each 0.2 mM dATP, dGTP, dCTP, dTTP
Primer solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
Probe solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
0.05 unit / μl rTth DNA polymerase (Toyobo)
0.01 μg / μl Anti-Tth antibody 0.05 unit / μl ReversetraAce (Toyobo)
(4) Reaction Using BioRad CFX96WELL DEEP, real-time PCR reaction was performed at the following temperature cycle. The fluorescence value was read in the extension step of 60 ° C. in the latter half 30 cycles.
42 ° C for 15 minutes (reverse transcription reaction),
95 ° C for 20 seconds,
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 10 cycles (PCR)
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 30 cycles (PCR)
(5) Result As a result, the Cq value of the G2 signal of each sample is shown in Table 3. Although the effective concentration differs depending on the sample, the guanidine thiocyanate concentration of the sample and the guanidine thiocyanate mixed solution is 0.33M or more, more preferably 0.6M or more, and it has a significant detection sensitivity compared to the condition where no mixing is performed. Improvement was observed. At this time, the concentration of guanidine thiocyanate in the RT-PCR reaction solution is 20 to 25 mM.

Example 3 FIG. Effect of mixing sample with urea (1) Sample preparation method Four specimens of human stool positive for Norovirus G2 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.
(2) Mixing method condition 1 with urea 2 M guanidine thiocyanate 0.5 μL and sample 0.5 μL
Condition 2 8 M urea 2.5 μL and sample 0.5 μL
Condition 3 3. 2 μL of 3.125M urea and 0.5 μL of sample
Condition 4 2 μL of 1M urea and 0.5 μL of sample
Condition 5 0.75 M urea 2 μL and sample 0.5 μL
Condition 6 0.5 M urea 2 μL and sample 0.5 μL
Condition 7 Sample 0.5 μl
Condition 8 2.5 μL of 8M urea and 1 μL of sample
Condition 9 7 M urea 2.5 μL and sample 1 μL
Condition 10 6.4 M urea 2.5 μL and sample 1 μL
Condition 11 6 M urea 2.5 μL and sample 1 μL
Condition 12 Sample 1 μL
(3) Reaction liquid 50 μl of the following reaction liquid was added to the mixed liquid.
1xBuffer (Blend Taq (registered trademark) attachment buffer (Toyobo))
0.3 M L-carnitine 1% Gelatin 1 mg / ml BSA
Each 0.2 mM dATP, dGTP, dCTP, dTTP
Primer solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
Probe solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
0.05 unit / μl rTth DNA polymerase (Toyobo)
0.01 μg / μl Anti-Tth antibody 0.05 unit / μl ReversetraAce (Toyobo)
(4) Reaction Using CFX96WELL DEEP manufactured by BioRad, real-time PCR reaction was performed at the following temperature cycle. The fluorescence value was read in the extension step of 60 ° C. in the latter half 30 cycles.
42 ° C for 15 minutes (reverse transcription reaction),
95 ° C for 20 seconds,
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 10 cycles (PCR)
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 30 cycles (PCR)
(5) Results Table 4 shows the Cq value of the G2 signal of each sample. Although the effective concentration differs depending on the sample, the urea concentration of the sample and the urea mixed solution is 2.5 M or more, more preferably 4.3 M or more, and a significant improvement in detection sensitivity is recognized as compared with the condition where no mixing is performed. At this time, the urea concentration in the RT-PCR reaction solution is 0.4 M or less.

Example 4 Comparison of Kaorotopic Method and Conventional Heat Treatment Method (1) Sample Norovirus positive human stool specimens 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.
(2) Detection by Chaotropic Method After mixing 8 M urea and 1 μL of each sample supernatant, 50 μl of the following reaction solution was added, and a real-time PCR reaction was performed using BioRad CFX96WELL DEEP at the following temperature cycle.
(2) -1 Reaction solution 1 × Buffer (Blend Taq (registered trademark) attachment buffer (Toyobo))
0.3 M L-carnitine 1% Gelatin 1 mg / ml BSA
Each 0.2 mM dATP, dGTP, dCTP, dTTP
Primer solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
Probe solution (Norovirus detection kit G1 / G2-probe detection-(Toyobo) attachment)
0.05 unit / μl rTth DNA polymerase (Toyobo)
0.01 μg / μl Anti-Tth antibody 0.05 unit / μl ReversetraAce (Toyobo)
(2) -2 The fluorescence value was read in the extension step at 60 ° C. in the latter half 30 cycles of the reaction cycle.
42 ° C for 15 minutes (reverse transcription reaction),
95 ° C for 20 seconds,
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 10 cycles (PCR)
95 ° C 1 sec-56 ° C 30 sec-60 ° C 25 sec 30 cycles (PCR)
(3) Norovirus detection kit G1 / G2-probe detection (Toyobo) was used as a detection detection reagent by the heat treatment method. The pretreatment liquid attached to the kit was added to 1 μL of each sample, and heat treatment was performed at 85 ° C. for 1 minute. After mixing the reaction solution, enzyme solution, primer solution, and probe solution attached to the kit, 45 μL each was added to each sample, and real-time PCR reaction was performed with the following temperature cycle.
The fluorescence value was read in the extension step at 60 ° C. in the latter half 30 cycles of the reaction cycle.
42 ° C for 5 minutes (reverse transcription reaction),
95 ° C for 30 seconds,
95 ° C 5 seconds-56 ° C 60 seconds 10 cycles (PCR)
95 ° C 5 seconds-56 ° C 60 seconds 30 cycles (PCR)
(4) Results In the probe solution (Norovirus detection kit G1 / G2-probe detection— (Toyobo) accessory), the FAM channel detects the internal control gene, the Cy5 channel detects Norovirus G1, and the ROX channel detects Norovirus G2. Here, Table 5 shows fluorescent dyes, Cq values, and type-specific determination results in which an increase in fluorescent signal was observed. With the method of the present invention in which no heat treatment was performed, the same results as in the conventional method in which heat treatment was performed were obtained.

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

Claims (19)

A method for examining an RNA virus having no envelope in a sample, the method comprising examining the presence or absence of a virus comprising the following steps without RNA purification from the sample or prior heat treatment of the sample.
(1) A step of mixing a sample and a reagent containing a chaotropic agent,
(2) adding a one-step RT-PCR reaction solution containing a reverse transcriptase and a thermostable DNA polymerase to the mixed solution;
(3) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel. The 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 the sample is feces. The method according to any one of claims 1 to 3, wherein the sample is a suspension suspended in water, physiological saline or a buffer. The method according to any one of claims 1 to 4, wherein the sample is a centrifugal supernatant of the suspension. The method according to claim 1 or 2, wherein the sample is a sample obtained by suspending a wiping test sample in the environment in water, physiological saline or a buffer, and concentrating the suspension. The method according to any one of claims 1 to 6, wherein the virus having no envelope is a norovirus. The method according to claim 7, wherein it is determined whether the norovirus is G1 type or G2 type. 9. The method according to claim 1, wherein the chaotropic agent is at least one selected from the group consisting of urea, thiourea, guanidine thiocyanate, guanidine hydrochloride, and arginine. The method according to any one of claims 1 to 9, wherein the thermostable DNA polymerase is selected from Taq, Tth or a variant thereof. The method according to any one of claims 1 to 10, wherein the origin of reverse transcriptase is selected from Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) or a variant thereof. . A one-step RT-PCR reaction solution is a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as “betaine-like quaternary ammonium”), 1 mg / ml or more of bovine serum The method according to any one of claims 1 to 11, comprising at least one selected from the group consisting of albumin, glycerol, glycol and gelatin. The method according to claim 12, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. A test kit for a virus having no envelope, comprising a reagent containing a chaotropic agent, a reverse transcriptase, a thermostable polymerase, and a one-step RT-PCR reaction solution. The kit according to claim 14, wherein the one-step RT-PCR reaction solution comprises at least one selected from the group consisting of betaine-like quaternary ammonium salts, 1 mg / ml or more of bovine serum albumin, glycerol, glycol and gelatin. . The kit according to claim 14 or 15, further comprising a primer pair corresponding to a detection region of a virus to be detected. The kit according to any one of claims 14 to 16, further comprising a hybridization probe corresponding to a detection region of a virus to be detected. The kit according to any one of claims 14 to 17, wherein the virus having no envelope is a norovirus. The kit according to claim 18, wherein it is determined whether the Norovirus is G1 type or G2 type.