JP2020115777A - Improved nucleic acid detection method - Google Patents

Abstract

To reduce the effect of substances contained in a biological sample that inhibit, over time, the nucleic acid amplification reaction, in particular, to suppress the degradation of RT-PCR efficiency even if the sample is not subjected to nucleic acid isolation and purification treatment or the like when feces are sampled.SOLUTION: The present invention relates to a method of examining the presence or absence of a target RNA in a sample, the method including the following steps: (1) adding a One-enzyme system 1-step RT-PCR reaction solution comprising a divalent cation and a thermostable DNA polymerase that are adjusted for a final concentration of 2.5 mM or more in the reaction solution to a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment; and (2) examining the presence or absence of the target RNA in the sample by a 1-step RT-PCR reaction after sealing the reaction container.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting RNA by nucleic acid amplification. More specifically, the reaction solution of reverse transcription polymerase chain reaction (RT-PCR) of one enzyme system is added to the sample without pretreatment including isolation (separation and purification) of nucleic acid from the sample and heating of the sample. Relates to the detection of RNA by adding. The method of the present invention can detect RNA in, for example, a stool sample, a blood sample, an environmental wipe sample, and the like. The present invention can be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

The nucleic acid amplification method is a technology that amplifies several copies of a target nucleic acid to a level at which it can be visualized, that is, hundreds of millions of copies or more, and is applied not only in the field of life science research but also in the medical field such as gene diagnosis and clinical tests, or in food and environment. It is also widely used in microbiological tests.
A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) Annealing of a primer to a template single-stranded DNA, and (3) of the primer using a DNA polymerase. This is a method of amplifying a target nucleic acid in a sample by setting three cycles of extension as one cycle and repeating this cycle. Annealing and extension may be performed in two steps at the same temperature.

The sample to be tested by the nucleic acid amplification method varies depending on the purpose. In the case of gene diagnosis targeting genomic DNA, it is a sample that has low invasiveness and can be easily collected, such as oral mucosal cells and blood. For detection of DNA or RNA such as bacteria or viruses that cause infectious diseases, samples in which causative microorganisms may be present are urine, sputum, feces, blood, nasal fluid, vaginal secretions, and the like. The food hygiene inspection is food or stool or urine collected from a food handler, the environmental hygiene inspection is soil or river water, environmental water such as rainwater or seawater, and wipes of manufacturing facilities.

Since such a sample also contains a large amount of substances other than many nucleic acids, it is usually necessary to separate and purify the nucleic acids in the sample in order to perform PCR efficiently. There are various methods for separating and purifying nucleic acids, but basically, it is composed of a step of separating a nucleic acid and other substances into a solid phase and a liquid phase and performing solid-liquid separation. For example, there are a method in which the nucleic acid is made into a solid phase by ethanol precipitation and separated by centrifugation, and a method in which the nucleic acid is hybridized to solid-phase beads and separated by centrifugation or a magnetized substance. However, these purification methods involving separation of the sample are complicated and time-consuming, and there is a risk that decomposition and contamination may occur during the operation. In addition, when the nucleic acid content in the sample is low, it may not be possible to recover the amount required for the amplification reaction. For these reasons, there has been a demand for a method of bringing a sample into a reaction solution and amplifying a target nucleic acid without going through a purification step involving separation of the sample.

Therefore, in recent years, a method has been studied in which a target nucleic acid is simply extracted from a biological sample such as feces by a pretreatment, and then the sample is not separated and purified, but detected by a PCR method (Non-Patent Document 1). However, in this case, a PCR reaction inhibitor such as a polysaccharide contained in the stool sample is brought in in order to omit the purification of the nucleic acid accompanying the separation of the sample, and the PCR reaction is affected to a considerable extent.

Measures for reducing these effects have been studied so far with respect to the composition of the pretreatment agent, PCR reaction solution, and the like (Patent Document 1). For example, it has been reported that PCR inhibition when detecting spiked DNA in a stool sample is improved by the addition of betaine, BSA, T4 gene 32 protein, and a protease inhibitor (Non-Patent Document 2). .. Furthermore, when detecting RNA by RT-PCR, a quaternary ammonium salt (betaine, L-carnitine, etc.) having a structure in which three methyl groups are added to amino acids, bovine serum albumin, glycerol, glycol, and It has been found that by combining all or part of gelatin, the PCR inhibition by feces can be eliminated (Patent Document 2).

However, there are some cases in which PCR does not proceed efficiently even when the sample is subjected to such treatment, and further improvement has been demanded.

JP, 2015-119656, A JP, 2017-131164, A

J. Virol. Methods, 163, 2010, pp. 282-286. J. Clin. Microbiol. , Volume 38, 2000, pp. 4643-4470.

As a result of various studies by the present inventors, after adding the RT-PCR reaction solution to an untreated sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment, when the RT-PCR reaction is started after a certain period of time, PCR is started. We have gained a new finding that has not been known so far, that the efficiency is significantly reduced. Then, the present inventor has further studied this decrease in PCR efficiency, and when feces are used as an example of a biological sample, the feces influence the divalent cations (particularly manganese ions) in the reaction solution over time. It was found for the first time that the nucleic acid amplification reaction may not proceed efficiently by doing so. The present invention solves such a problem, and it takes about 15 minutes after addition of the RT-PCR reaction solution to an untreated sample that has not undergone nucleic acid separation/purification treatment or heat treatment after a certain period of time has passed. It is an object of the present invention to provide a method and the like that can efficiently perform nucleic acid detection by an RT-PCR reaction performed by using an untreated sample even after a certain amount of time).

As a result of studying a method for solving the above-mentioned problems, the present inventor separated the sample by setting the divalent cation concentration in the RT-PCR reaction solution added to the untreated sample to a predetermined concentration or more. The present invention has been completed based on the finding that it can be used for an amplification reaction without accompanying nucleic acid purification or prior heat treatment.

The representative invention of the present application is as follows.
[Item 1] A method for inspecting the presence or absence of target RNA in a sample, which comprises the following steps:
(1) Includes a divalent cation and a thermostable DNA polymerase adjusted so that the final concentration in the reaction solution is 2.5 mM or more for a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment A step of adding one enzyme system one-step RT-PCR reaction solution, and
(2) A step of inspecting the presence or absence of target RNA in a sample by a 1-step RT-PCR reaction after sealing the reaction container.
[Item 2] The method according to Item 1, wherein the divalent cation is a manganese ion.
[Item 3] The method according to Item 1 or 2, wherein the final concentration of the divalent cation in the reaction solution is adjusted to 3 mM or more.
[Item 4] The method according to any one of Items 1 to 3, wherein the final concentration of the thermostable DNA polymerase in the reaction solution is 4.2 ng/μL or less.
[Item 5] The method according to any one of Items 1 to 4, wherein the steps (1) and (2) are performed in the same container.
[Item 6] The method according to any one of Items 1 to 5, wherein after the reaction container is sealed in the step (2), the 1-step RT-PCR reaction is carried out without opening and closing the lid even once.
[Item 7] In step (2), heat treatment is performed before and/or during the PCR cycle reaction to expose nucleic acid in the sample and/or to perform hot start PCR in the PCR reaction. Item 7. The method according to any one of Items 1 to 6, including.
[Item 8] The method according to Item 7, wherein the heat treatment is performed at 60° C. or higher and for 1 second or longer.
[Item 9] The method according to any one of Items 1 to 8, wherein the sample is a fecal sample.
[Item 10] The method according to any one of Items 1 to 9, wherein the sample is a sample suspension suspended in water, physiological saline, or a buffer solution.
[Item 11] The method according to any one of Items 1 to 10, wherein the sample is a centrifugal supernatant of a sample suspension.
[Item 12] The method according to any one of Items 1 to 11, wherein the target RNA is a nucleic acid derived from an RNA virus having no envelope.
[Item 13] The method according to Item 12, wherein the RNA virus is a Norovirus.
[Item 14] The method according to Item 13, wherein it is possible to determine whether the Norovirus is GI type or GII type.
[Item 15] The method according to any one of Items 1 to 14, wherein the thermostable DNA polymerase is a DNA polymerase belonging to Family A.
[Item 16] The thermostable DNA polymerase is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Taq, Tth, Z05, and variants thereof. 15. The method according to any one of 15.
[Item 17] The method according to any one of Items 1 to 16, wherein the RT-PCR reaction solution further comprises a primer pair corresponding to the detection region of the target RNA to be detected.
[Item 18] The method according to any one of Items 1 to 17, wherein the RT-PCR reaction solution further contains a hybridization probe corresponding to the detection region of the target RNA to be detected.
[Item 19] In a one-enzyme system one-step RT-PCR reaction performed using a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment, RT-PCR is performed over time by mitigating the influence of contaminants in the sample. A method for enhancing reaction stability, which comprises allowing a thermostable DNA polymerase and a divalent cation having a final concentration of 2.5 mM or more to coexist in an RT-PCR reaction solution.
[Item 20] An enzyme-based 1-step RT-PCR composition for use in the method according to any one of Items 1 to 19, wherein the final concentration in the RT-PCR reaction solution is 2.5 mM or more. A one-enzyme system one-step RT-PCR reaction composition comprising a divalent cation adjusted as described above and a thermostable DNA polymerase.
[Item 21] A kit for inspecting the presence or absence of target RNA in a sample, which comprises the composition for one-step RT-PCR reaction of one enzyme system according to Item 20.

According to the present invention, it is possible to reduce the time and cost for separating and purifying nucleic acid and heat-treating a sample prior to the nucleic acid amplification reaction. Further, it is possible to reduce the risk that may occur when nucleic acid is separated and purified or the sample is heat-treated in advance, that is, the risk of contamination of other samples due to sample loss or carryover. In particular, the effect becomes remarkable in an inspection in which a large number of specimens using feces as samples are processed.

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

The method for testing the presence or absence of target RNA in a sample of the invention is characterized in that it comprises at least the following steps:
(1) Includes a divalent cation and a thermostable DNA polymerase adjusted so that the final concentration in the reaction solution is 2.5 mM or more for a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment A step of adding one enzyme system one-step RT-PCR reaction solution, and
(2) A step of inspecting the presence or absence of target RNA in a sample by a 1-step RT-PCR reaction after sealing the reaction container.
According to the above-described inspection method of the present invention, a sample that has not been subjected to a nucleic acid separation/purification treatment and/or a heat treatment as a pretreatment (in the present specification, this is used unless otherwise specified) When performing an RT-PCR reaction performed by providing "an untreated sample" or "a sample that has not been pretreated" or the like, a certain period of time has elapsed after mixing the sample with the RT-PCR reaction solution. Even after that, a decrease in PCR efficiency can be suppressed and stable test results can be obtained. Thereby, for example, in a test in which it is desired to collectively process a large amount of samples (for example, a food hygiene control test such as norovirus), the mixing process of the sample from each subject and the RT-PCR reaction solution is performed. Even after some time elapses until the RT-PCR reaction of each mixed solution after performing, there is an advantage that the test result can be stably obtained.

In the above inspection method of the present invention, the RT-PCR reaction is not a two-step method in which the reverse transcription reaction and the PCR reaction are performed in separate steps, but the reverse transcription reaction and the PCR reaction are performed continuously or in parallel in one step. It is characterized in that it is performed by a one-step method. In the inspection method of the present invention, the steps (1) and (2) can be performed in the same container, or the steps (1) and (2) can be performed in separate containers. The steps (1) and (2) are performed in the same container from the viewpoint that the risk of the reaction mixture liquid due to the transfer of the container and the risk of contamination can be reduced, and further it can be performed more easily. It is preferably carried out. That is, it is preferable not to transfer all or part of the mixed solution to another container between the steps (1) and (2). Furthermore, in step (2), it is preferable that the lid of the reaction container is not opened and closed after the reaction container is sealed. By not opening and closing the lid after sealing as described above, the risk of contamination can be more effectively reduced, and more accurate inspection results can be obtained.

The test method of the present invention is characterized in that the one-enzyme one-step RT-PCR reaction solution used in the step (1) contains 2.5 mM or more of a divalent cation. The divalent cation is not particularly limited as long as the effects of the present invention are exhibited, but typical ions include, for example, magnesium ion and manganese ion, and manganese ion is particularly preferable. When using a magnesium salt, for example, a magnesium salt such as magnesium chloride, magnesium sulfate, or magnesium acetate may be added to the RT-PCR reaction solution. When using a manganese salt, for example, a manganese salt such as manganese chloride, manganese sulfate, or manganese acetate may be added to the RT-PCR reaction solution. When magnesium ions and manganese ions are added in the form of salts such as magnesium salts and manganese salts, they are added to the RT-PCR reaction solution so that the final concentration in the RT-PCR reaction solution will be 2.5 mM or more. .. Preferably, the final concentration in the reaction solution is 3 mM or more, more preferably the final concentration in the reaction solution is 3 to 10 mM, and further preferably the final concentration in the reaction solution is 3 to 5 mM, Above all, it is preferable to add it at a concentration adjusted so that the final concentration in the reaction solution is 3 to 3.5 mM. Since the one-enzyme system one-step RT-PCR reaction solution contains a divalent cation at such a final concentration, stable high PCR can be achieved even when an untreated sample is allowed to coexist for a certain period of time. It becomes possible to perform inspection efficiently.

Examples of biological samples used in the present invention include feces (feces, rectal feces), vomit, saliva, blood, etc., but are not particularly limited, and any biological sample can be used. Is. Among them, the present invention is particularly fecal (fecal excretion, rectal feces) because it contains a large amount of contaminants and thus tends to cause a decrease in PCR reactivity over time after mixing with an RT-PCR reaction solution. Useful for the detection of RNA from

The present invention is characterized by detecting RNA as it is, or without extracting the nucleic acid from the biological sample and performing purification involving separation of the sample. That is, the present invention is characterized in that it is not necessary to isolate RNA from these biological samples with a commercially available RNA purification kit, or to perform heat treatment in advance to expose RNA from the viral structure. .. In other words, the untreated sample used in the present invention may be a sample that has been subjected to nucleic acid extraction without separation and purification, as long as it is not subjected to heat treatment. Here, the sample that has been subjected to nucleic acid extraction without separation and purification means to expose the nucleic acid in the sample, for example, by destroying the capsid, envelope, etc. in the sample, and encapsulated in these Extracting the exposed nucleic acid and exposing it (however, the capsid and the fragment of the envelope remaining after the destruction are not removed). In the present invention, good PCR efficiency can be maintained and stable test results can be obtained even with a sample prepared by omitting such labor for isolation and purification. The nucleic acid extraction treatment that does not involve such separation and purification can be performed prior to the step (1).

In a particular embodiment, in the step (2), heat treatment is carried out before and/or during the PCR cycle reaction to expose the nucleic acid in the sample and/or hot start in the PCR reaction. PCR can be performed. By performing the heat treatment in the step (2), specifically, the pathogenic microorganism (eg, virus) intended to be detected can be crushed and the nucleic acid of the pathogenic microorganism (eg, virus) can be exposed in the sample. it can. Further, when the RT-PCR reaction solution contains an enzyme or the like that can function in the hot start PCR method by performing the heat treatment in the step (2) as described above, such a hot start enzyme can be activated. It will be possible. There is no particular limitation on the heating temperature and the heating time as long as the heat treatment can achieve the intended purpose, but the heat treatment may be 60° C. or higher and 1 second or longer, preferably 70° C. , 30 seconds or more, more preferably 80° C., 30 seconds or more, particularly preferably 85° C., 30 seconds or more.

In the test method of the present invention, the biological sample may be directly subjected to detection, but from the viewpoint of reducing the influence of contaminants on the reaction and facilitating more stable test results, water, physiological saline, or a buffer solution is used. A sample in which the sample is suspended in a liquid is preferable. Furthermore, in the case of a sample containing a lot of contaminants such as feces, the supernatant may be used after centrifugation. Alternatively, filter filtration may be performed. The buffer used in the suspension is not particularly limited, but preferably Hanks buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, tricine buffer and the like. To be The pH of the buffer solution is also not particularly limited as long as the effect of the present invention is exhibited, but from the viewpoint that the effect of the present invention can be obtained more reliably, it is preferably neutral to alkaline, more preferably pH=7. -11, more preferably pH=9-11.

As a sample of another aspect of the present invention, a wiping test sample can be mentioned. A wiping test is useful for elucidating the pollution route and grasping the pollution situation of the facility environment. In the present invention, the wiping test is not particularly limited, but it is, for example, a sample obtained by wiping the corresponding section or equipment with a cotton swab or the like, eluting with water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitation or the like. .. Specific examples of the wiping test include “improvement of the norovirus test method for wiping samples” (http://idsc.nih.go.jp/iasr/32/382/dj3824.html) and the like. The method is not particularly limited, and widely includes methods according to this. Examples of the wiping points include cutting boards, kitchen knives, dish towels, tableware and other cooking utensils, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, faucets such as bathrooms, cooks' hands and fingers, and bathrooms. , Toilets, washbasins, handrails, living rooms, etc. Moreover, although not a wiping test, it can be applied to a concentrated sample of a sewage sample as an environmental test.

The biological sample may include a step of inactivating a substance capable of inhibiting the nucleic acid amplification reaction, such as a protein, before being added to the RT-PCR reaction solution. When the protein is inactivated in this way, in the present invention, the inactivated protein is subjected to the RT-PCR reaction without being separated and purified from the sample. Examples of the step of inactivating include, but are not limited to, alkali treatment, treatment with an organic solvent, and treatment combining a plurality of these treatments. On the other hand, these treatments must be performed under conditions such that the nucleic acid retains its function as a template for amplification. Specifically, the pH during the alkali treatment is preferably 9 to 11, more preferably 10 to 11.

The sample that has undergone the above treatment is added to the RT-PCR reaction solution without undergoing purification that involves separation of the sample. If a precipitate or the like is generated by the above treatment, centrifugation may be performed. In this case, there is an advantage that it is not necessary to separate the sample if the above-mentioned treatment is performed using a container applicable to centrifugation.

The target RNA to be detected by the test method of the present invention (this may be referred to as target RNA etc.) is not particularly limited. In general, tests for detecting RNA or the like derived from infectious microorganisms (bacteria or viruses that cause infectious diseases) from biological samples such as feces are for the purpose of identifying the cause of food poisoning, identifying the infection route, and preventing it. Has been widely performed. For example, inspection of norovirus is often performed by a method of detecting a gene by RT-PCR method using feces as a sample. Feces are composed of intestinal bacteria, intestinal epithelial cells, food-derived substances, and the like, and are known to contain a large amount of contaminants such as polysaccharides. The present invention suppresses the decrease in PCR efficiency even in a sample containing a large amount of such contaminants and in which inhibition of the RT-PCR reaction is concerned, and it is possible to obtain good test results.

More specifically, an example of a test target for testing RNA in feces includes RNA derived from a pathogenic virus, but is not particularly limited. Examples of these viruses include non-enveloped RNA viruses that do not have an envelope derived from a lipid bilayer membrane. Such non-enveloped RNA viruses include Astroviridae viruses (eg Astrovirus); Caliciviridae viruses (eg Sapovirus, Norovirus); Picornaviridae viruses (eg Hepatitis A virus, Echovirus, Enterovirus, coxsackievirus, poliovirus, rhinovirus); hepeviridae virus (eg, hepatitis E virus); reoviridae virus (eg, rotavirus). Although not limited, the detection method of the present invention is preferably useful for detection of caliciviridae virus and reoviridae virus, more preferably for detection of norovirus, sapovirus, rotavirus, It is especially useful for detecting norovirus. Among them, the test method of the present invention is preferable because it can exert a high effect in the test of GII type norovirus. Further, in the case of norovirus, it can be applied as a means for discriminating between GI type and GII type.

As an example of the composition of the RT-PCR reaction solution that can be used in the present invention, a pair of two kinds of primers in which one primer is complementary to the DNA extension product of the other primer, dNTP, thermostable DNA polymerase, It may contain a divalent cation, a monovalent ion, a buffer solution and the like adjusted to a final concentration of 2.5 mM or more in the reaction solution. More specifically, as an example of the composition of the RT-PCR reaction solution that can be used in the present invention, a pair of two primers in which one primer is complementary to the DNA extension product of the other primer, dNTP, heat resistance It may contain DNA polymerase, magnesium ion adjusted to have a final concentration of 2.5 mM or more in the reaction solution, BSA, a nonionic surfactant, a buffer solution and the like.

In the inspection method of the present invention, the pair of two types of primers can be appropriately selected and designed according to the target RNA and used, and is not particularly limited. Further, when the target RNA to be targeted is assumed to be a subtype, it may be a degenerate primer. Examples of the two pairs of primers that can be used in the present invention include, for example, two pairs of primers designed to detect norovirus RNA, which is one of RNA viruses having no envelope, and sapovirus RNA. It may be a pair of two primers designed according to the above, a pair of two primers designed to detect rotavirus RNA, and the like. As a primer for detecting norovirus RNA, for example, the primer (SEQ ID NOS: 1 to 5; SEQ ID NOS: 1 and 2 are Norovirus G1 types) described in the Notification of the Safety and Safety Division, Safety Department, Pharmaceutical and Food Bureau, Ministry of Health, Labor and Welfare , SEQ ID NOs: 3 to 5 are primers for detecting Norovirus G2 type), but are not limited thereto.

In another aspect, the present invention may use a so-called multiplex RT-PCR reaction solution containing two or more pairs of the above primers. The two or more pairs of primers that can be included in the case of the multiplex RT-PCR reaction solution can be any set of primer pairs, for example, a primer pair for detecting norovirus RNA and a rotavirus RNA are detected. For detecting norovirus RNA, a pair of primers for detecting norovirus RNA and a pair of primers for detecting sapovirus RNA, and a pair of primers for detecting GI type norovirus RNA and a type GII norovirus RNA A preferable example is a set of primer pairs for the purpose. Furthermore, when performing a multiplex RT-PCR reaction, not all detection targets need to be RNA, and for example, a multiplex RT-PCR reaction for detecting target RNA and target DNA as targets. May be In this case, the multiplex RT-PCR reaction solution contains a primer pair for detecting target RNA (for example, a primer pair for detecting norovirus RNA) and a primer pair for detecting target DNA (for example, DNA virus or A set of primer pairs for detecting DNA from enterobacteria may be included depending on the purpose.

As an example of the amplification reaction, a biological sample is added to the above reaction composition without purification accompanied by separation of the sample, and as PCR cycle reactions, (1) DNA denaturation, (2) primer annealing, ( 3) A method of performing thermal cycling of primer extension, or (1) DNA denaturation, (2) thermal cycle of performing primer annealing and primer extension simultaneously (shuttle PCR), but not limited thereto. ..

The 1-step RT-PCR reaction solution added to the mixed solution contains a thermostable DNA polymerase. Examples of the DNA polymerase include, but are not limited to, Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and variants thereof belonging to Family A DNA polymerase. .. The present invention is characterized in that a thermostable DNA polymerase having reverse transcriptase activity is used to perform the one-enzyme system one-step RT-PCR reaction. Here, the DNA polymerase having reverse transcriptase activity also means a DNA polymerase having both the ability to convert RNA into cDNA and the ability to amplify DNA. As a DNA polymerase having a reverse transcriptase activity, a DNA polymerase derived from Thermus aquaticus (Taq), a DNA polymerase derived from Thermus thermophilus HB8 (Tth), a DNA polymerase derived from Thermus sp Z05 (Z05), and a Thermotoga maritima derived. Polymerase (Tma), Bacillus caldotenax-derived DNA polymerase (Bca), Bacillus stearothermophilus-derived DNA polymerase (Bst) and the like can be mentioned. Good. Further, a mutant of DNA polymerase (KOD) derived from Thermococcus kodakaraensis, which has reverse transcription activity is known (for example, RTX: reverse transcription xenopolymerase), and the present invention has such reverse transcriptase activity. It is not limited as long as it is a thermostable DNA polymerase having both. Particularly preferred is a DNA polymerase having a reverse transcriptase activity selected from the group consisting of Taq, Tth, Z05 and variants thereof, and among them, Tth DNA polymerase which is a DNA polymerase having a high reverse transcriptase activity is preferred. ..

The thermostable DNA polymerase used in the present invention is not particularly limited as long as it has a thermostability that can sufficiently function even at a high temperature in a PCR reaction cycle, but for example, even if heat treatment is performed at 85° C. for 1 minute or more. It is preferable that the heat resistance is such that the enzyme activity does not decrease more than half. In a specific embodiment, the 1-step RT-PCR reaction solution used in the present invention preferably further contains an anti-DNA polymerase antibody in order to enhance the effect of suppressing non-specific reaction. In a further specific embodiment, a thermostable DNA polymerase having a thermosensitive chemical modification at the active site of the enzyme can also be used. By further including an anti-DNA polymerase antibody or using a thermostable DNA polymerase that has been subjected to a heat-sensitive chemical modification, the enzymatic activity of the DNA polymerase is suppressed during the reverse transcription reaction, and hot start PCR is performed. Since it can be applied to, it is possible to detect with higher specificity, which is preferable.

The total amount of thermostable DNA polymerase in the one-enzyme one-step RT-PCR reaction solution used in the present invention is not particularly limited as long as the effects of the present invention are exhibited. Since the present invention can exhibit high PCR efficiency, the total amount of thermostable DNA polymerase may be small. For example, the final concentration in the reaction solution is 10 ng/μL or less, preferably 5 ng/μL or less, and more preferably It can be 4.2 ng/μL or less, and for example, it may be possible to obtain a good test result even if it is less than 4.2 ng/μL. The lower limit of the final concentration in the reaction solution is not particularly limited, but may be 0.01 ng/μL or more, for example. By using the thermostable DNA polymerase at such a concentration, nonspecific nucleic acid amplification can be suppressed. The amount of DNA polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be estimated from a safety data sheet (SDS). When a protein such as BSA is included, it is desirable to calculate by the latter method.

In a specific embodiment, the 1-step RT-PCR reaction solution used in the present invention comprises a thermostable DNA polymerase as described above, a buffer, an appropriate salt, and a final concentration of 2.5 mM or more in the reaction solution. Adjusted to generate divalent cations (eg, magnesium salt or manganese salt), deoxynucleotide triphosphate (dNTP), primer pair corresponding to detection target region of viral RNA to be detected, reverse transcription For the purpose of suppressing the reaction inhibition during the reaction, an RNA degrading enzyme inhibitor and, if necessary, an additive and the like may be contained.

The buffer that can be used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, Bicine, and the like. Examples thereof include sulfuric acid, hydrochloric acid, acetic acid, It may be a buffer solution adjusted to pH 6 to 9, more preferably pH 7 to 8 with phosphoric acid or the like. The concentration of the buffering agent to be added is not particularly limited as long as the effects of the present invention are exhibited, but for example, it can be used at about 10 to 200 mM, more preferably about 20 to 150 mM.

A salt (for example, a salt solution) is preferably added to the 1-step RT-PCR reaction solution used in the present invention in order to obtain suitable ionic conditions for the RT-PCR reaction. Suitable salts include, but are not limited to, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like. These appropriate salts may be added to the 1-step RT-PCR reaction solution used in the present invention in the form of a salt solution dissolved in a solvent such as water.

The dNTP that can be used in the present invention is not particularly limited, but may be dNTP containing dATP, dCTP, dGTP, and dTTP in equal amounts, for example, dATP, dCTP, dGTP, and dTTP are 0.1 to 0.5 mM. Within the range of dNTPs, preferably dATP, dCTP, dGTP, and dTTP each containing about 0.2 mM. It is also possible to take prophylactic measures against cross contamination by using dUTP instead of and/or as a part of dTTP.

Further, as an additive contained in the 1-step RT-PCR reaction solution used in the present invention, 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 salt" Ammonium)), 0.5 mg/ml or more of bovine serum albumin, glycerol, glycol, and gelatin.

Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine) and L-carnitine. 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 the betaine-like quaternary ammonium salt is a compound with stable positive and negative charges in the molecule, and it is believed that it exhibits surfactant-like properties and destabilizes the viral structure. Moreover, it may be possible to facilitate nucleic acid amplification of DNA polymerases. The preferred concentration of the betaine-like quaternary ammonium salt is such that the final concentration in the RT-PCR reaction solution is preferably 0.1 M to 2 M, more preferably 0.2 M to 1.2 M.

The bovine serum albumin that can be contained in the 1-step RT-PCR reaction solution can be adjusted so that the final concentration in the reaction solution is 0.5 mg/ml or more. For a sample containing a lot of contaminants such as feces, it is possible to perform better detection by adjusting the final concentration of bovine serum albumin in the reaction solution to preferably 1 mg/ml or more.

Gelatin that can be contained in the 1-step RT-PCR reaction solution is derived from the skin and bones of animals such as cows and pigs, the tendons, or the scales and skins of fish, and is considered to contribute to the stabilization of PCR enzymes. The concentration used is preferably such that PCR amplification is stabilized while fluorescence detection is not hindered. The final concentration in the reaction solution is preferably 0.1 to 5%, and more preferably adjusted to be the final concentration in the reaction solution of 0.5 to 2%. The origin of gelatin is not particularly limited, but those derived from fish are preferable to those derived from bovine or porcine because jelly strength is low and handling of the reaction solution is good.

Furthermore, the 1-step RT-PCR reaction solution used in the present invention can be used in combination with a substance known in the art to promote RT-PCR. Examples of the RT-PCR promoter useful in the present invention include glycerol, polyol, protease inhibitor, 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, ectoin, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethyl acetate Examples thereof include, but are not limited to, ammonium (TMAA), polyethylene glycol, Triton X-100 (Triton X-100), Triton X-114 (Triton X-114), Tween 20 (Tween 20), Nonidet P40, Briji 58 and the like. To further reduce the 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.

In certain embodiments, the detection methods of the invention may further employ at least one labeled hybridization probe or double-stranded DNA binding fluorescent compound. By using such a probe, the analysis of the nucleic acid amplification product can be monitored not by ordinary electrophoresis but by monitoring the fluorescent signal, and the analysis labor is reduced. Furthermore, it is not necessary to open the reaction vessel, and it is possible to further reduce the risk of contamination. For example, it is also possible to identify the virus subtype by labeling each hybridization probe corresponding to the subtype of the target viral RNA to be detected 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, but are not limited thereto.

Examples of the hybridization probe 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 beacons (US Pat. No. 5,118,801), FRET hybridization probes (WO 97/46707 pamphlet, WO 97/46712 pamphlet). , International Publication No. 97/46714 pamphlet) and the like. As the nucleotide sequence of the probe for detecting norovirus, the sequences (SEQ ID NOS: 6 to 9; SEQ ID NOS: 6 and 7 are Norovirus G1 type) described in the notification of the Safety and Safety Division, Safety Department, Pharmaceutical and Food Bureau, Ministry of Health, Labor and Welfare SEQ ID NOs: 8 and 9 are probes for detecting Norovirus G2 type), but are not limited thereto. Furthermore, when the target RNA targeted is expected to be a subtype, a degenerate sequence may be included.

As described above, in the one-enzyme one-step RT-PCR reaction performed by using a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment, the present inventor ensures that the final concentration in the reaction solution is 2.5 mM or more. By allowing the divalent cation and the thermostable DNA polymerase to coexist in the PCR, the PCR efficiency does not decrease even after a certain time has elapsed since the untreated sample and the RT-PCR reaction solution are allowed to coexist, and stable test results can be obtained. It finds that it can be obtained.

Therefore, from another aspect, the present invention is to alleviate the influence of contaminants in a sample in a one-enzyme one-step RT-PCR reaction carried out using a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment. Is a method for enhancing the stability of RT-PCR reaction over time, characterized in that a bivalent cation having a final concentration of 2.5 mM or more and a thermostable DNA polymerase coexist in the RT-PCR reaction solution. A method is also provided.
The type and concentration of the divalent cation and the thermostable DNA polymerase, the sample, etc. used in the above method for enhancing the stability are the same as the above-mentioned method for inspecting the presence or absence of the target RNA in the sample.

Further, from another viewpoint, the present invention provides a composition for one-step 1-step RT-PCR reaction for use in the method for inspecting the presence or absence of target RNA in a sample as described above, which comprises a RT-PCR reaction solution. Also provided is a composition for one-step 1-step RT-PCR reaction, which comprises a divalent cation adjusted to a final concentration of 2.5 mM or more, and a thermostable DNA polymerase.
The type and concentration of the divalent cation and thermostable DNA polymerase used in the above composition, the sample, and the like are also the same as in the above-described method for inspecting the presence or absence of target RNA in the sample.

Yet another aspect of the present invention may be a kit for use in the method for examining the presence or absence of target RNA in a sample as described above. Specifically, the present kit is characterized by containing the above-described composition for one-enzyme system one-step RT-PCR reaction. The kit may include a package insert describing the procedure of the inspection method of the present invention.
The type and concentration of the divalent cation and thermostable DNA polymerase used in the kit for inspecting the presence or absence of the target RNA in the sample described above also depend on the presence or absence of the target RNA in the sample. The method is the same as the inspection method.

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

Test Example 1. Effect of a lapse of time after mixing feces and reaction solution (1) Sample One human feces sample, which was negative for both Norovirus G1 and G2, was suspended at 10% (weight %). This suspension was centrifuged at 12,000 rpm for 5 minutes and used as an untreated fecal suspension. Further, a part of the untreated stool suspension was taken, heated at 98° C. for 5 minutes, and used as a stool suspension subjected to heat treatment.
(2) Reaction Solution The reaction solution having the composition shown below was used as a basic composition to evaluate the influence of feces on the nucleic acid amplification reaction.
Reaction solution (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
50 mM Mn(OAc) ₂ (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
10x primer solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
10x probe solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
4.2 ng/μl rTth DNA polymerase (Toyobo)
0.01 μg/μl Anti-Tth antibody (3) reaction The above reaction solutions were mixed so that the final concentration of Mn (manganese ion) was 2.63 mM, and the mixture was dispensed in 19 μl portions. 1 μl of (a) pure water, (b) untreated stool suspension, and (c) heat-treated stool suspension were added to each of these reaction solutions. Then, the mixture was allowed to stand at room temperature for 15 minutes, and then 1250 copies of synthetic RNAs of Norovirus G1 and G2 (each N=4) were added. Using this, LightCycler (registered trademark) 96 System manufactured by Roche was used to perform a real-time PCR reaction in a temperature cycle described in RNA-direct ^™ Realtime PCR Master Mix (Toyobo).
(4) Results The probe solution (Norovirus detection kit G1/G2-fast probe detection-supplied by Toyobo) detects an internal control gene in the FAM channel, Norovirus G1 in the Cy5 channel, and Norovirus G2 in the ROX channel. Here, the Cq values of G1 and G2 RNAs are shown in Table 1. Also, n. d. Indicates that the nucleic acid could not be detected. In the presence of feces, a Cq value delay of about 4 for Norovirus G1 and about 8 for Norovirus G2 was confirmed as compared to the absence of feces. Therefore, in the presence of feces, it becomes difficult to detect 2 ⁴ =16 and 2 ⁸ =256 times, respectively, when detecting the same amount of nucleic acid. In addition, it was confirmed that in the stool suspension to which heat treatment was applied, the Cq value of Norovirus G2 was about 2, that is, 2 ² =4 times easier to detect than the untreated stool suspension. Based on these results, it is clear that the stool suspension after standing at room temperature for 15 minutes inhibits the RT-PCR reaction, and it is untreated stool suspension as compared with the heat-treated stool suspension. Was found to have a greater effect of the inhibition.

Test example 2. Effect of immediate start of reaction after mixing feces and reaction solution (1) Sample One human feces sample negative for both Norovirus G1 and G2 was suspended at 10% (wt%). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used. This supernatant was diluted with pure water so that the concentration was 1, 2.5, 5, 10 times.
(2) Reaction Solution The reaction solution having the composition shown below was used as a basic composition to evaluate the influence of feces on the nucleic acid amplification reaction.
Reaction solution (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
50 mM Mn(OAc) ₂ (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
10x primer solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
10x probe solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
4.2 ng/μl rTth DNA polymerase (Toyobo)
0.01 μg/μl Anti-Tth antibody (3) reaction The above reaction solutions were mixed so that the final concentration of Mn was 2.63 mM, and the mixture was dispensed in 19 μl portions. To these reaction solutions, 1250 copies of synthetic RNA of Norovirus G1 and G2 (N=4) or 1 μl of pure water was added. After transferring this reaction solution to an 8-strip tube, 1 μl of a dilution series (1, 2.5, 5, 10 times) of the untreated stool suspension was attached to the wall surface of the 8-strip tube (each N=2). , And mixed by centrifugation just before RT-PCR. In addition, a real-time PCR reaction was carried out using a LightCycler (registered trademark) 96 System manufactured by Roche in a temperature cycle described in RNA-direct ^™ Realtime PCR Master Mix (Toyobo).
(4) Results Table 2 shows the Cq values of G1 and G2 RNAs. As a result, compared to Test Example 1 in which the undiluted solution of the stool suspension was left for 15 minutes after addition, the reaction solution in which the undiluted solution of the stool suspension was mixed immediately before the reaction was about 2 for Norovirus G1 and 6 for Norovirus G2. It became smaller. Therefore, in the case of detecting the same amount of nucleic acid, it becomes difficult to detect 2 ² =4, 2 ⁶ =64 times, respectively, depending on the lapse of time after adding the stool suspension to the reaction solution. In addition, since the delay of the Cq value decreased when the dilution ratio of the stool suspension was increased, it is clear that the feces contain a component that inhibits the reaction. From this result, it was found that there was feces in which the inhibition became greater with time after mixing the stool suspension and the reaction solution.

Test example 3. Identification of Components of Reaction Solution Affected by Feces (1) Samples The following samples were prepared.
(Stool sample suspension)
One sample of human feces negative for both Norovirus G1 and G2 was suspended at 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.
(2) Reaction Solution The reaction solution having the composition shown below was used as a basic composition to evaluate the influence of feces on the nucleic acid amplification reaction.
Reaction solution (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
50 mM Mn(OAc) ₂ (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
10x primer solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
10x probe solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
4.2 ng/μl rTth DNA polymerase (Toyobo)
0.01 μg/μl Anti-Tth antibody (3) reaction Prepare 19 μL of reaction solution containing Mn at a final concentration of 2.63 mM (reaction solution A) and 18 μL of reaction solution not containing Mn (reaction solution B), and suspend in stool 1 μl of the liquid was added, and the mixture was left at room temperature for 15 minutes. Then, to the reaction solution B, 1 μL of 50 mM Mn(OAc) ₂ was added so that the final concentration of Mn was 2.63 mM. Furthermore, 1250 copies of synthetic RNAs of Norovirus G1 and G2 (each N=2) were added to the reaction solution having a total volume of 20 μL. Immediately after the addition of Mn and RNA, a real-time PCR reaction was carried out using a LightCycler (registered trademark) 96 System manufactured by Roche in a temperature cycle described in RNA-direct ^™ Realtime PCR Master Mix (Toyobo).
(4) Results Table 3 shows Cq values of G1 and G2 RNAs. Also, n. d. Indicates that the nucleic acid could not be detected. Comparing the reaction solution A and the reaction solution B, the Cq value of the reaction solution B for both Norovirus G1 and G2 was about 1 to 2 smaller. Therefore, when the same amount of nucleic acid is detected, it is considered that the reaction solution B is easier to detect 2 ¹ to ^{2 2} =2 to 4 times. In the reaction solution A, Mn and feces were allowed to coexist for 15 minutes, and in the reaction solution B, the real-time PCR reaction was carried out immediately after the addition of Mn. Therefore, since it was difficult to detect nucleic acid when Mn and feces in the reaction solution were allowed to coexist for a certain period of time, it was found that the component of the reaction solution affected by feces was Mn.

Test example 4. Optimization of Mn concentration in a system containing feces (1) Samples The following samples were prepared.
(Stool sample suspension)
One sample of human feces negative for both Norovirus G1 and G2 was suspended at 10% (% by weight). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.
(2) Reaction Solution The reaction solution having the composition shown below was used as a basic composition to evaluate the influence of feces on the nucleic acid amplification reaction.
Reaction solution (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
50 mM Mn(OAc) ₂ (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
10x primer solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
10x probe solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
4.2 ng/μl rTth DNA polymerase (Toyobo)
0.01 μg/μl anti-Tth antibody (3) reaction The above reaction solutions were mixed so that the final concentration of Mn was 1.58, 2.63, 3.16, 3.68, 4.74 mM, and 19 μl each Dispensed. To these reaction solutions, 1 μl of a stool suspension was added and left at room temperature for 15 minutes. 1250 copies of synthetic RNA of Norovirus G1 and G2 (each N=4) were added to the reaction solution having a total volume of 20 μL. Norovirus G1, G2 synthetic RNA 6250 (N=1), 1250 (N=1), 250 (N=1), 50 (N=2), 25 (N=2), 0 (N=1) copies Was added. Using this, LightCycler (registered trademark) 96 System manufactured by Roche was used to perform a real-time PCR reaction in a temperature cycle described in RNA-direct ^™ Realtime PCR Master Mix (Toyobo).
(4) Results Table 3 shows the Cq values of G1 and G2 RNAs. Also, n. d. Indicates that the nucleic acid could not be detected. In Norovirus G1, the Cq value in the presence of feces decreased by about 1 to 2 as the Mn concentration increased. Therefore, when detecting the same amount of nucleic acid, it is considered that detection is facilitated by 2 ¹ to ^{2 2} =2 to 4 times. On the other hand, in Norovirus G2, it can be said that the Cq value is about ¹ to ^{2 at} 3 mM, that is, 2 ¹ to ^{2 2} = 2 to 4 times easier to detect than the Mn concentration of 2.5 mM. Further, since the detection sensitivity was improved by about 5 times with 250 copies at 2.5 mM and 50 copies at 3 mM, it was concluded that detection in the presence of feces was facilitated by the increase in Mn concentration. To be In this test example, a delay in the Cq value was not confirmed by increasing the Mn concentration, even though 15 minutes had elapsed after adding the stool suspension to the reaction solution. Based on these results, it was found that by increasing the Mn concentration, the inhibition of feces can be remarkably alleviated, and the influence of the passage of time after mixing the feces and the reaction solution can be sufficiently suppressed.
From the above results, in the system containing feces, the final Mn concentration in the RT-PCR reaction solution was 2.5 mM or more, preferably 3 mM or more, and the improvement in Cq value equivalent to that in the system containing no feces was observed.

Test example 5. Detection of Specimens by One-Enzyme System 1-Step RT-PCR (1) Preparation of Samples 3 specimens of human feces were suspended so as to have a concentration of 10% (wt %). This suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used.
(2) Reaction Solution The reaction solution having the composition shown below was used as a basic composition to evaluate the influence of feces on the nucleic acid amplification reaction.
Reaction solution (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
50 mM Mn(OAc) ₂ (attached to RNA-direct ^™ Realtime PCR Master Mix (Toyobo))
10x primer solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
10x probe solution (Norovirus detection kit G1/G2-fast probe detection-(Toyobo) attachment)
4.2 ng/μl rTth DNA polymerase (Toyobo)
0.01 μg/μl Anti-Tth antibody (3) reaction Mix the above reaction solutions so that the final concentration of Mn will be 2.63, 3.16, and 3.68 mM, and dispense in 19 μl aliquots to suspend feces. 1 μl of the solution was added. This was subjected to a real-time PCR reaction using the LightCycler (registered trademark) 96 System manufactured by Roche at the following temperature cycle.
90°C for 1 minute 60°C for 5 minutes (reverse transcription reaction)
95°C 1 second-54°C 10 seconds 50 cycles (PCR)
(4) Results G1 and G2 Table 5 shows the Cq values of norovirus in feces. Also, n. d. Indicates that the nucleic acid could not be detected. All of the samples 1 to 3 used this time were positive for Norovirus G2. Since the Cq value decreases as the Mn concentration increases, it can be determined that the detection of the sample is facilitated. From these results, it was confirmed that increasing the Mn concentration in the one-enzyme system one-step RT-PCR reaction solution facilitates detection of norovirus in feces.

INDUSTRIAL APPLICABILITY The present invention is suitably used in the field of molecular biology research, and further in the field of tests for clinical tests and food hygiene control. In particular, the present invention can save the labor of isolating and purifying nucleic acids, for example, in a test in which it is desired to collectively process a large amount of samples (for example, a food hygiene control test such as norovirus). In addition, it is very useful because it is possible to obtain stable test results even after some time elapses after mixing such an untreated sample with the RT-PCR reaction solution.

Claims (21)

A method for examining the presence or absence of target RNA in a sample, which comprises the following steps:
(1) Includes a divalent cation and a thermostable DNA polymerase adjusted so that the final concentration in the reaction solution is 2.5 mM or more for a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment A step of adding one enzyme system one-step RT-PCR reaction solution, and
(2) A step of inspecting the presence or absence of target RNA in a sample by a 1-step RT-PCR reaction after sealing the reaction container. The method according to claim 1, wherein the divalent cation is a manganese ion. The method according to claim 1 or 2, wherein the final concentration of the divalent cation in the reaction solution is adjusted to 3 mM or more. 4. The method according to claim 1, wherein the final concentration of the thermostable DNA polymerase in the reaction solution is 4.2 ng/μL or less. The method according to claim 1, wherein the steps (1) and (2) are performed in the same container. The method according to any one of claims 1 to 5, wherein after the reaction vessel is sealed in the step (2), the 1-step RT-PCR reaction is carried out without opening and closing the lid even once. In step (2), comprising performing a heat treatment before and/or during the PCR cycle reaction to expose the nucleic acid in the sample and/or to perform hot start PCR in the PCR reaction. 7. The method according to any one of 1 to 6. The method according to claim 7, wherein the heat treatment is performed at a temperature of 60° C. or higher for 1 second or longer. The method according to claim 1, wherein the sample is a fecal sample. The method according to any one of claims 1 to 9, wherein the sample is a sample suspension suspended in water, physiological saline or a buffer solution. The method according to claim 1, wherein the sample is a centrifugal supernatant of a sample suspension. The method according to any one of claims 1 to 11, wherein the target RNA is a nucleic acid derived from an RNA virus having no envelope. The method according to claim 12, wherein the RNA virus is Norovirus. The method according to claim 13, wherein it is possible to determine whether the norovirus is GI type or GII type. The method according to any one of claims 1 to 14, wherein the thermostable DNA polymerase is a DNA polymerase belonging to Family A. 16. The thermostable DNA polymerase is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Taq, Tth, Z05, and variants thereof, any one of claims 1 to 15. The method described in crab. The method according to any one of claims 1 to 16, wherein the RT-PCR reaction solution further comprises a primer pair corresponding to the detection region of the target RNA to be detected. The method according to claim 1, wherein the RT-PCR reaction solution further contains a hybridization probe corresponding to the detection region of the target RNA to be detected. In the one-enzyme 1-step RT-PCR reaction performed by using a sample that has not been subjected to nucleic acid separation/purification treatment or heat treatment, RT-PCR reaction stability over time is improved by mitigating the influence of contaminants in the sample. A method of increasing, wherein a bivalent cation having a final concentration of 2.5 mM or more and a thermostable DNA polymerase coexist in an RT-PCR reaction solution. A one-enzyme one-step RT-PCR reaction composition for use in the method according to claim 1, wherein the final concentration in the RT-PCR reaction solution is adjusted to 2.5 mM or more. A composition for one-step enzyme-based one-step RT-PCR reaction, characterized in that it comprises the above-mentioned divalent cation and a thermostable DNA polymerase. A kit for inspecting the presence or absence of target RNA in a sample, which comprises the composition for one-step RT-PCR reaction of one enzyme system according to claim 20.