JP2022191442A - Method for suppressing non-specific nucleic acid amplification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for specifically performing DNA amplification from a template RNA through a one-step RT-PCR method while suppressing non-specific nucleic acid amplification.

SOLUTION: In one embodiment, the present invention provides a method for amplifying a nucleic acid from a sample through one-step RT-PCR, the method comprising the steps of: (1) preparing a one-step RT-PCR reaction solution containing a sample, an anionic polymer, and (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity; and (2) carrying out a one-step RT-PCR reaction after sealing a reaction vessel. The concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention relates to methods for amplifying nucleic acids. For example, the present invention provides compositions and methods useful for amplification of nucleic acids from ribonucleic acid (RNA) templates, specifically compositions and methods for nucleic acid amplification from reverse transcription reactions using anionic polymers. Regarding the method. More specifically, it relates to nucleic acid amplification by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the presence of anionic polymers. The method of the present invention specifically detects RNA virus-derived RNA in, for example, throat swabs, saliva samples, sputum samples, fecal samples, blood samples, environmental swab samples, etc., and biological RNA in biological samples, etc. enables. 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 a few copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more. It is also widely used in microbiological examinations, etc. A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR consists of (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) conversion of the primer using a DNA polymerase. This is a method of amplifying a target nucleic acid in a sample by repeating three steps of elongation as one cycle. In some cases, annealing and extension are performed at the same temperature in two steps.

When analyzing RNA, reverse transcription (RT), which converts template RNA into cDNA, is performed as a preceding stage of this PCR. This is called RT-PCR. This RT-PCR is roughly divided into (1) 2-step RT-PCR in which RT and PCR are performed discontinuously, and (2) 1-step RT-PCR in which RT and PCR are performed continuously. Among RT-PCRs, one-step RT-PCR is preferred for genetic testing and virus testing because of high throughput and avoiding contamination due to opening and closing of the reaction vessel during the reaction.

The detection sensitivity of one-step RT-PCR is greatly related to the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase. Until now, in order to improve the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase, the use of amino acid mutants with improved enzymatic activity (Patent Document 1), various additives, etc. has been studied ( Patent document 2). In one-step RT-PCR in a two-enzyme system containing reverse transcriptase and DNA polymerase, it is also known that reverse transcriptase itself inhibits PCR. A method of adding is also known (Patent Document 3).

In addition to reverse transcription efficiency of reverse transcriptase and DNA synthesis efficiency of DNA polymerase, non-specific gene amplification affects detection sensitivity. Non-specific gene amplification refers to amplification of nucleic acid sequences other than the target gene in PCR. PCR is susceptible to contaminants such as dyes, proteins and sugars, and it is known that contaminants inhibit the reaction or cause non-specific nucleic acid amplification. Therefore, it is usually necessary to purify the nucleic acid in advance when nucleic acid is amplified from a sample. However, purification of nucleic acids is complicated and time-consuming, and there is a risk of contamination during the operation. In addition, when the content of the target nucleic acid in the sample is low, it may not be possible to recover the target nucleic acid. For these reasons, there is a need for a simple and efficient method of amplifying a target nucleic acid by suppressing non-specific reactions even when an unpurified sample is used.

In addition, multiple primer sets can be used to detect two or more target regions at the same time, to save reagents, equipment, time, etc., or to limit the amount of template samples. A so-called multiplex PCR method, a PCR method for simultaneous amplification in a reaction solution, may be performed. However, even in multiplex PCR, primer dimers and non-specific reactions are likely to occur, and it has sometimes been difficult to amplify the target nucleic acid with high sensitivity. Therefore, even when performing multiplex PCR, there is a demand for development of a method for suppressing non-specific reactions and amplifying target nucleic acids simply and efficiently.

Furthermore, in one-step RT-PCR, all reactions are performed in the same reaction vessel, so non-specific amplification occurs, resulting in deoxynucleotide triphosphate (dNTP ) will be depleted. As a result, the target gene cannot be amplified in a sufficient amount, resulting in a decrease in detection sensitivity.

A number of methods have been reported to reduce non-specific amplification in one-step RT-PCR. For example, optimization of amplification conditions (increase annealing temperature, decrease number of cycles, decrease amount of enzyme, change type of enzyme, decrease dNTP concentration, decrease Mg concentration or Mn concentration, decrease template DNA concentration, etc.), It is known to change the design of primers, use hot start methods using antibodies or aptamers, and use modified oligonucleotides (PNA, LNA, etc.) that are said to increase specificity.

However, although such a method may be effective in suppressing non-specific amplification, it is often not sufficiently effective or even suppresses specific amplification. Furthermore, since it is necessary to examine RT-PCR conditions and primer design, it is often troublesome. Therefore, it has been desired to develop a simple and further method capable of suppressing non-specific amplification in one-step RT-PCR.

International Publication No. 2018096961A pamphlet Japanese Patent Application Laid-Open No. 2018-000138 Japanese Patent No. 4777497 JP 2012-24039 A Japanese Patent Application Laid-Open No. 2017-023110 JP 2016-182112 A

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

The present invention has been made in view of the above-mentioned prior art, and is a simple method for nucleic acid amplification from a template RNA by one-step RT-PCR, but it can be used, for example, for nucleic acid amplification and multiplex PCR from unpurified samples. It is an object of the present invention to provide a further useful means capable of effectively suppressing non-specific nucleic acid amplification even in the case of nucleic acid amplification at 100°C and enabling highly sensitive and specific detection of a target nucleic acid.

In view of the above circumstances, the present inventors have conducted intensive research and found that the use of an anionic polymer can suppress non-specific nucleic acid amplification in one-step RT-PCR, resulting in highly sensitive detection of target nucleic acids. The inventors have found that specific detection is possible, and have completed the present invention.

The representative invention of the present application is as follows.
[Claim 1] A method for amplifying nucleic acids from a sample by one-step RT-PCR, comprising the following steps;
(1) preparing a one-step RT-PCR reaction solution comprising a sample, an anionic polymer, and (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase with reverse transcription activity; and (2) After sealing the reaction vessel, performing a one-step RT-PCR reaction;
and wherein the concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more.
[Item 2] The nucleic acid amplification method according to Item 1, wherein the sample used in the step (1) is a biological sample, an environmental sample, or a cell-derived sample that has not undergone a purification step.
[Claim 3] The sample used in the step (1) is saliva, sputum, pharyngeal swab, nasal swab, gargle, feces, lung aspirate, cerebrospinal fluid, gargle, tears, cultured cells, culture Item 3. The nucleic acid amplification method according to Item 1 or 2, wherein the sample is at least one selected from the group consisting of supernatant, swab test sample, soil sample, and sewage sample.
[Claim 4] The sample used in the step (1) is suspended in at least one selected from the group consisting of water, physiological saline, buffer, and sputazyme enzyme solution, or a suspension thereof. Item 4. The nucleic acid amplification method according to any one of Items 1 to 3, which is a centrifugal supernatant or concentrate.
[Item 5] The nucleic acid amplification method according to any one of Items 1 to 4, wherein the sample used in step (1) is a sample that may contain an RNA virus.
[Item 6] The nucleic acid amplification method according to any one of Items 1 to 5, wherein two or more target regions are specifically amplified in one one-step RT-PCR reaction solution.
[Item 7] The nucleic acid amplification method according to Item 6, wherein the two or more target regions are target regions in the genomic RNA of an RNA virus that may be contained in the sample.
[Item 8] The nucleic acid amplification method according to any one of items 5 to 7, wherein the RNA virus is an enveloped RNA virus.
[Item 9] The enveloped RNA virus is a coronavirus family virus, a flaviviridae virus; a togaviridae virus; an orthomyxoviridae virus; a rhabdoviridae virus; a bunyaviridae virus; Item 9. The method for amplifying nucleic acid according to Item 8, wherein the virus is at least one selected from the group consisting of family viruses.
[Claim 10] At least one enveloped RNA virus selected from the group consisting of SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2 coronavirus 10. The method for amplifying nucleic acid according to Item 8 or 9.
[Item 11] The method for amplifying nucleic acid according to any one of items 5 to 7, wherein the RNA virus is a non-enveloped RNA virus.
[Item 12] The non-enveloped RNA virus is at least one selected from the group consisting of Caliciviridae virus, Astroviridae virus, Picornaviridae virus, Hepeviridae virus, and Reoviridae virus. 12. The nucleic acid amplification method according to item 11.
[Item 13] The anionic polymer is obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. Item 13. The nucleic acid amplification method according to any one of items 1 to 12, wherein the polymer is a
[Item 14] The anionic polymer is polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, carrageenan, heparin, chondroitin sulfate, keratan from sulfuric acid, hyaluronic acid, heparan sulfate, chondroitin, dermatan sulfate, polyvinylsulfonic acid, polyvinylphosphonic acid, polystyrenesulfonic acid, polyacrylic acid, polyacrylic acid/sulfonic acid copolymers, and polyacrylic acid/maleic acid copolymers Item 14. The nucleic acid amplification method according to any one of items 1 to 13, wherein the method is at least one selected from the group consisting of:
[Item 15] The method for amplifying nucleic acid according to any one of items 1 to 14, wherein the anionic polymer has an average molecular weight of 1,000 to 5,000,000.
[Item 16] The nucleic acid amplification method according to any one of items 1 to 15, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth and variants thereof.
[Item 17] The origin of the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV), and variants thereof. Item 17. The nucleic acid amplification method according to any one of items 1 to 16.
[Item 18] The nucleic acid amplification method according to any one of Items 1 to 17, wherein the one-step RT-PCR reaction solution in step (1) further contains one or more primer pairs corresponding to the target region.
[Item 19] The nucleic acid amplification method according to any one of items 1 to 18, wherein the one-step RT-PCR reaction solution in step (1) further contains a hybridization probe corresponding to the target region.
[Item 20] The one-step RT-PCR reaction solution in the step (1) contains a quaternary ammonium salt (hereinafter referred to as "betaine-like quaternary ammonium") having a structure in which three methyl groups are added to the amino group of an amino acid. Item 20. The nucleic acid amplification method according to any one of Items 1 to 19, further comprising at least one selected from the group consisting of bovine serum albumin, glycerol, glycol, gelatin, and polar organic solvents.
[Item 21] The method for amplifying nucleic acid according to Item 20, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine.
[Item 22] The nucleic acid amplification method according to any one of Items 1 to 21, wherein the concentration of the anionic polymer in the one-step RT-PCR reaction solution in the step (1) is 0.0025 to 0.05%. .
[Claim 23] A method for suppressing non-specific amplification, characterized in that an anionic polymer is allowed to coexist in the one-step RT-PCR reaction solution in nucleic acid amplification by one-step RT-PCR from a sample that has not undergone a purification step.
[Item 24] The method for suppressing non-specific amplification according to Item 23, wherein an anionic polymer is allowed to coexist in an amount of 0.001% or more in the one-step RT-PCR reaction solution.
[Claim 25] A method for nucleic acid amplification by one-step RT-PCR for specifically amplifying two or more target regions from a sample in one reaction solution, characterized in that an anionic polymer is allowed to coexist in the reaction solution. A nucleic acid amplification method.
[Item 26] A reagent containing an anionic polymer, which is used for suppressing non-specific amplification in nucleic acid amplification by one-step RT-PCR from a sample that has not undergone a purification step.
[Item 27] The reagent according to Item 26, which is adjusted so that the concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more.
[Item 28] A reagent for use in the nucleic acid amplification method by one-step RT-PCR reaction according to any one of Items 1 to 25, which contains an anionic polymer.
[Item 29] A kit for use in a nucleic acid amplification method by one-step RT-PCR reaction, containing the reagent according to any one of Items 26 to 28.

According to the present invention, non-specific nucleic acid amplification in the one-step RT-PCR reaction can be suppressed by allowing the anionic polymer to coexist in the reaction solution, thereby producing a specific amplification product of the target nucleic acid intended to be amplified. Since the amount can be increased, it may be possible to improve the detection sensitivity.

For example, according to the present invention, samples that may contain RNA viruses such as SARS-nCOV-2 that occurred in 2019 can also exhibit excellent effects. In addition, according to the present invention, blood, feces (excretion, rectal stool), vomit, urine, sputum, lymph, plasma, ejaculate, lung aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, gargle Nucleic acids in samples containing large amounts of contaminants, such as biological samples containing fluid, saliva, tears, environmental swabs, cultured cells or supernatants, even without prior purification of these samples It is also possible to detect RNA viruses such as coronaviruses contained in the virus with high sensitivity. The present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

Effect of anionic polymers on RT-PCR inhibition by saliva samples (3 μL). Effect of anionic polymers on RT-PCR inhibition by saliva samples (8 μL). FIG. 4 shows the effect of anionic polymers on sensitivity to RT-PCR inhibition by saliva specimens.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited to these. It should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified.
Also, all non-patent documents and patent documents mentioned in this specification are incorporated herein by reference. In the present specification, "~" means "more than or equal to or less than". For example, "X to Y" in the specification means "X or more and Y or less". Also, "and/or" in this specification means either or both. Also, in this specification, it should be understood that the singular terms also include the plural terms unless stated otherwise.

One embodiment of the present invention is a method of nucleic acid amplification by one-step RT-PCR reaction using RNA contained in a sample as a template, by performing a one-step RT-PCR reaction in the presence of an anionic polymer. This is a method for amplifying nucleic acids while suppressing specific nucleic acid amplification. Here, non-specific nucleic acid amplification means that a nucleic acid sequence other than the target sequence of template RNA is synthesized by one-step RT-PCR.

In general, when a nucleic acid (eg, gene) is amplified using RNA as a template, a reverse transcription reaction (also called RT reaction) is first performed to convert the template RNA into cDNA, and the resulting cDNA is amplified by PCR reaction. One-step RT-PCR refers to a method of nucleic acid amplification from template RNA by performing the above-described RT reaction and PCR reaction consecutively or in parallel. The one-step RT-PCR includes a two-enzyme one-step RT-PCR reaction in which the RT reaction and the PCR reaction are sequentially performed using two different enzymes (reverse transcriptase and DNA polymerase), A one-enzyme one-step RT-PCR or the like is known in which the RT reaction and the PCR reaction are performed consecutively or in parallel using a single enzyme (a DNA polymerase having reverse transcriptase activity such as Tth DNA polymerase). there is The invention can be practiced in any one-step RT-PCR.

One-step RT-PCR preferably performs all reactions in the same reaction vessel. When one-step RT-PCR is carried out in the same reaction vessel in this way, if non-specific nucleic acid amplification occurs, substrates such as deoxynucleotide triphosphate (dNTP), etc., which are required for target nucleic acid amplification that should occur originally, may occur. is depleted, the target nucleic acid cannot be amplified in a sufficient amount, and there is a risk of causing a decrease in detection sensitivity. Therefore, the method of the present invention, which can suppress non-specific nucleic acid amplification in a one-step RT-PCR reaction, can suppress such decrease in target nucleic acid detection sensitivity. Therefore, the nucleic acid amplification method of the present invention can be called a method for suppressing non-specific amplification, and can also be called, for example, a method for increasing the production amount of a specific nucleic acid amplification product, a method for improving specific amplification of a target nucleic acid, and the like. can.

Examples of template RNA include biological samples such as tissues, body fluids or secretions collected from living organisms (e.g., biological tissues, body fluids or secretions that may contain pathogenic microorganisms such as viruses), environmental samples, cells (e.g., cultured cells etc.), and may be, for example, RNA extracted from biological samples, environmental samples or cells, or RNA that has been further purified after extraction processing (e.g., ethanol precipitation, column purification, etc.). It may be purified RNA treated by any purification means known in the art), etc., or an RNA-containing sample that has not undergone such an RNA purification step, or any RNA.

In one preferred embodiment, the present invention is a method for nucleic acid amplification by one-step RT-PCR from a biological sample, environmental sample, or cell-derived sample that has not undergone a purification step. Here, the term “purification” refers to separating the target nucleic acid RNA in the sample from tissues, cell walls, and other contaminants that may be contained in various samples. or separation of nucleic acids using an ion-exchange resin, a glass filter, or a reagent having a protein-aggregating action. Therefore, in the present invention, the "sample that has not undergone a purification process" refers to various samples (eg, biological samples) themselves, liquid samples diluted with a solvent such as water, and solid samples diluted with water or the like. Examples include those obtained by adding to a solvent and crushing by applying heat. For example, when the nucleic acid to be amplified exists in a tissue, cell, outer shell such as an envelope or capsid of a sample such as an organ or cell, these tissues, cells, outer shells, etc. are used to extract the nucleic acid. The act of destroying (destruction by physical treatment, extraction act such as destruction using a surfactant, etc.) does not correspond to the purification referred to in the present invention. Further, the act of diluting the sample obtained by the above method or various samples themselves with water, buffer solution, etc. does not correspond to the purification referred to in the present invention.

As one form, when extracting RNA from a tissue, the site of the tissue is not particularly limited, and any tissue may be used. A method for extracting RNA from a tissue usually includes a cell isolation step using a cell strainer, trypsin treatment, or the like, but the method is not particularly limited.
Alternatively, when extracting RNA from a cell or outer shell such as an envelope or capsid, any kind of cell may be used without particular limitation. For example, the number of cells can be appropriately adjusted using a cell sorter. For example, RNA extracted from a small number of cells (eg, 1-100, preferably 1-10, more preferably 1 or 2, more preferably 1) can also be used. A method for extracting RNA from cells or the like usually uses a cell lysing composition or a pretreatment composition containing a cell lysing agent or the like (for example, a pretreatment composition for extracting RNA from a virus or the like). , the step of lysing cells and the like. Examples of cell lysing agents include surfactants and chaotropic agents. Surfactants include anionic surfactants (e.g. sodium dodecyl sulfate, sodium cholate, sodium deoxycholate), cationic surfactants (e.g. cetyltrimethylammonium bromide), nonionic surfactants (e.g. , octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate), and zwitterionic surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]). -1-propanesulfonic acid). Chaotropic agents include, for example, urea and lithium salts such as lithium perchlorate. Cell lysing agents are usually dissolved in water (preferably nuclease-free water) and used in the form of an aqueous solution. Cell lysing compositions may include proteases (eg, protease K), RNase inhibitors, combinations of two or more thereof.
The composition for cell lysis or the composition for pretreatment may or may not contain the anionic polymer. When RNA extracted from cells using the cell lysis composition or pretreatment composition containing the anionic polymer is used as the template RNA, the composition for use in the one-step RT-PCR reaction additionally contains the above-mentioned There is an advantage that it is not necessary to add an anionic polymer.

As another form, when RNA is extracted from a biological sample, the biological sample may be, for example, feces (excretion, rectal stool), urine, vomit, saliva, sputum, pharyngeal swab, nasal swab, gargle, lung aspirate. cerebral spinal fluid, tear fluid, nasal discharge, blood (whole blood), plasma, serum, etc., but not limited to, and can be used for all substances derived from living organisms, depending on the purpose. can be selected as appropriate. In a preferred embodiment, the biological sample includes saliva, sputum, pharyngeal swab, nasal swab, gargle, feces, lung aspirate, cerebrospinal fluid, gargle, tears, cultured cells, culture supernatant, and the like. to use. The target template RNA may be biologically derived RNA, or may be RNA derived from microorganisms, viruses, or the like contained in a biological sample. In addition, the sample may be directly subjected to detection without going through a purification step, or may be diluted with water, physiological saline, or a buffer solution to reduce the influence of contaminants on the reaction and to obtain more stable test results. It may be a sample in which the sample is suspended in. Examples of the buffer include, but are not limited to, Hank's buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and tricine buffer. In the case of a highly viscous biological sample (eg, a sample containing highly viscous sputum), the sample may be treated with a sputazyme enzyme solution, although not particularly limited.

As yet another aspect, the invention can be practiced to perform nucleic acid amplification from RNA in environmental samples such as swab samples. For example, the present invention can be used to detect target RNA from swab test samples, soil samples, sewage samples, etc. as described above, but is not particularly limited, and any sample derived from the environment can be used. Wiping tests are useful for clarifying the route of contamination by viruses and bacteria and for grasping the contamination status of facility environments. In the present invention, the wiping test is not particularly limited, but for example, the relevant section or equipment is wiped with a cotton swab or the like, eluted in water or a buffer solution, and concentrated by polyethylene glycol (PEG) precipitation. . Specific examples of swabbing test procedures include "Improvement of swabbing test method for norovirus" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). It is not particularly limited, and includes a wide range of similar methods. Examples of areas to be wiped include kitchen utensils such as cutting boards, kitchen knives, dish towels, tableware, refrigerator handles, toilets, bathroom doorknobs, washrooms, kitchens, toilets, bathroom faucets, cooks' hands and fingers, and bathrooms. , toilets, washrooms, handrails, living rooms, and other facilities. Moreover, although it is not a wiping test, it can also be applied to a concentrated sewage sample as an environmental test.

In the present invention, the concentration of template RNA added to the one-step RT-PCR reaction solution is, for example, 1 to 10000 pg/μL, preferably 10 to 1000 pg/μL, more preferably 1 to 100 pg/μL. Not limited.

The target RNA virus of the present invention may be either a non-enveloped RNA virus derived from a lipid bilayer membrane or an enveloped RNA virus. In certain preferred embodiments, the present invention is highly effective in suppressing non-specific amplification in nucleic acid amplification from enveloped RNA viruses.

Specifically, non-enveloped RNA viruses (also referred to as "non-enveloped RNA viruses", etc.) include Astroviridae viruses (e.g., Astrovirus); Caliciviridae viruses (e.g., Sapovirus, Norovirus); Lunaviridae viruses (e.g., hepatitis A virus, echovirus, enterovirus, coxsackievirus, poliovirus, rhinovirus); Hepeviridae viruses (e.g., hepatitis E virus); Reoviridae viruses (e.g., rotavirus), etc. Although not limited, it is preferably useful for detecting Caliciviridae viruses and Reoviridae viruses, more preferably useful for detecting noroviruses, sapoviruses, and rotaviruses, and still more preferably It is useful for detecting norovirus and rotavirus, and particularly useful for detecting norovirus.

Enveloped RNA viruses (also referred to as "enveloped RNA viruses") include Flaviviridae viruses (e.g., hepatitis C virus, Japanese encephalitis virus, Zika virus, swine fever virus); Togaviridae viruses (e.g., rubella virus coronaviruses (e.g. SARS coronavirus, MERS coronavirus, SARS-nCOV-2 coronavirus); orthomyxoviridae viruses (e.g. influenza virus); rhabdoviridae viruses (e.g. rabies virus) ); Bunyaviridae viruses (e.g., Crimea-Congo fever virus, hantavirus); Paramyxoviridae viruses (e.g., measles virus, human respiratory syncytial virus); Filoviridae viruses (e.g., Ebola virus), etc. , is not particularly limited. From the viewpoint that the high effect of the present invention can be more reliably obtained, it is preferably useful for detecting Coronaviridae viruses, and more preferably SARS coronavirus, MERS coronavirus, and SARS-nCOV-2 coronavirus. It is useful for detection, especially for detection of SARS-nCOV-2 coronavirus (also called SARS-CoV-2).

Coronaviruses are causative viruses that cause respiratory infections including colds, and about 10 to 35% of common colds are said to be caused by coronaviruses. Mutant viruses are also known to occur, and rarely SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, novel coronavirus infectious disease (COVID-19) coronavirus (SARS) -nCOV-2) are known to cause fatal serious respiratory diseases. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronaviruses is important in clinical diagnosis, food hygiene inspections, environmental inspections, and the like. Useful.

Conventionally, methods for detecting pathogens of coronaviruses have been developed using electron microscopy, immunological antigen detection methods using ELISA, or viral gene detection methods using nucleic acid amplification technology. Among these test methods, nucleic acid amplification technology that can detect coronaviruses with high sensitivity is widely used, and several related technologies have been developed (for example, Non-Patent Document 1, Patent Document 2, Patent Document 4).

In particular, for the mutated coronavirus SARS-nCOV-2, which was confirmed to occur in Wuhan City, Hubei Province, China in 2019, a test method using nucleic acid amplification technology will be established as soon as the analysis of the virus genome RNA is completed. (For example, Non-Patent Document 3, Non-Patent Document 4). Also in Japan, a method for detecting SARS-nCOV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases (Non-Patent Document 5). In these techniques, detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample. The extraction and purification steps of viral RNA, especially the purification step, are complicated and require a lot of working time. In recent years, in the detection of influenza virus, a method is known in which a virus extract obtained by mixing a throat swab sample with a pretreatment liquid containing a water-soluble organic solvent and a surfactant is used as a sample (Patent Document 5, Patent Document 6). Also, K. Kang et al. reported that highly pathogenic North American porcine reproductive and respiratory syndrome virus RNA can be detected directly from porcine serum samples by RT-PCR (Non-Patent Document 6). In these techniques, by omitting the RNA extraction and purification steps, RT-PCR reaction inhibitors contained in the sample are brought into the reaction solution. RT-PCR reaction inhibitors vary greatly depending on the type of sample. For example, saliva samples carry large amounts of PCR reaction inhibitors such as polysaccharides and digestive enzyme RNase. In addition, it is known that virus inactivation and RNA extraction conditions vary greatly depending on the virus species, but the prior art literature does not mention any effect on coronaviruses (Patent Document 5). , Patent Document 6). Currently, coronaviruses, especially SARS-nCOV-2, can be rapidly detected from biological samples such as pharyngeal/nasal swabs, saliva, sputum, and stool samples, as well as environmental swabs by one-step RT-PCR without the need for RNA purification. It is desired to develop a method that can The present invention is particularly useful in these methods because it can suppress non-specific reactions and enable highly sensitive detection even in nucleic acid amplification from unpurified samples.

In the present invention, an anionic polymer is a polymer formed by polymerization of mainly anionic monomers. For example, the anionic polymer used in the present invention mainly polymerizes a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. and preferably a polymer obtained by polymerizing a sulfonic acid group as a monomer. Nucleic acid molecules such as RNA and DNA are also anionic polymers. Nucleic acid molecules contained in the sample that are different from the target can bind to reverse transcriptase or DNA polymerase in the reaction solution and cause non-specific nucleic acid amplification. While not wishing to be bound by theory, in the present invention, an anionic polymer capable of binding to reverse transcriptase or DNA polymerase is added to the reaction system to compete with the binding of the target to a different nucleic acid molecule. As a result, it is expected to have the effect of suppressing non-specific nucleic acid amplification.

The anionic polymer is not particularly limited as long as the effect of the present invention is exhibited, but typical examples include nucleic acid polymers (polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid), polysaccharides (carrageenan, heparin, chondroitin sulfate, keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin, dermatan sulfate), polyvinylsulfonic acid, polyvinylphosphonic acid, polystyrenesulfonic acid, polyacryl acids, polyacrylic acid/sulfonic acid copolymers, polyacrylic acid/maleic acid copolymers, and the like.

The anionic polymer may be in the form of a salt. For example, it may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt), or the like, or a hydrate salt. Alkali metal salts are preferred, sodium salts and potassium salts are more preferred, and sodium salts are even more preferred.

The average molecular weight of the anionic polymer is not particularly limited as long as the effects of the present invention are exhibited. In addition, in this specification, average molecular weight means weight average molecular weight. The average molecular weight of the anionic polymer depends on the molecular weight and the degree of polymerization of the monomers that are the constituent units. 000 or more. In addition, the upper limit of the average molecular weight of the anionic polymer is not particularly limited as long as the effect of the present invention is exhibited. can be:

The concentration of the anionic polymer in the one-step RT-PCR reaction solution (so-called final concentration in the reaction solution) is not particularly limited as long as the effect of the present invention is exhibited, but it is said that the high effect of the present invention can be reliably exhibited. From the point of view, it is preferably 0.001% (v/v%) or more. From the viewpoint that a higher non-specific reaction suppression effect can be expected, the concentration of the anionic polymer in the one-step RT-PCR reaction solution is preferably 0.002% or more, more preferably 0.0025% or more, and 0.005%. % or more is more preferable, and 0.008% or more is even more preferable. For example, it may be 0.01% or more, 0.02% or more, or 0.04% or more. The upper limit of the anionic polymer concentration in the one-step RT-PCR reaction solution is not particularly limited as long as the effect of the present invention is exhibited, but it can be, for example, 0.1% or less, preferably 0.05% or less. In addition, when the sample is purified or pretreated in advance, an anionic polymer is added to the reaction solution for the purification or pretreatment, and the reaction solution is added to the one-step RT-PCR reaction solution to perform nucleic acid amplification. When this is done, it is preferable to adjust the concentration of the anionic polymer to be brought into the 1-step RT-PCR reaction solution so that it falls within the above range.

One embodiment of the present invention is a method for nucleic acid amplification from a sample by one-step RT-PCR, comprising the steps of:
(1) preparing a one-step RT-PCR reaction solution comprising a sample, an anionic polymer, and (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase with reverse transcription activity; and (2) After sealing the reaction vessel, performing a one-step RT-PCR reaction;
and wherein the concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more.
The nucleic acid amplification method described above can suppress non-specific nucleic acid amplification and efficiently amplify the template RNA in the sample with high specificity.
Here, in the step (1), the reverse transcription reaction and the subsequent nucleic acid amplification reaction may be performed. Even a one-enzyme reaction system containing a thermostable DNA polymerase with reverse transcription activity can perform a one-step RT-PCR reaction. Prior to the step (1), a step of purifying RNA from a sample, a step of pretreating the sample by mixing it with a treatment solution containing an acidic or alkaline solution, an organic solvent, a surfactant, etc., and a step of heat-treating the mixture. A process or the like may be implemented, or may not be implemented. The anionic polymer may be contained in the reaction solution for performing the one-step RT-PCR reaction. The anionic polymer may be contained in the 1-step RT-PCR reaction solution, or when the sample is purified or pretreated in advance, the anionic polymer is added to the treatment solution that has been purified or pretreated. It may be formulated and carried into the reaction system when the treatment solution is added to the one-step RT-PCR reaction.

The steps (1) and (2) are preferably carried out in the same container. That is, between steps (1) and (2), it is preferable not to transfer all or part of the mixture to another container. Furthermore, in step (2), it is preferable not to open and close the lid of the reaction vessel after sealing the reaction vessel.

The RT-PCR cycle in step (2) includes: 1. reverse transcription reaction;2. It consists of two steps of PCR. A heat treatment step may be included before and after each step to activate the hot start enzyme. The temperature of the reverse transcription reaction in 1 is determined by the reverse transcription activity of the thermostable DNA polymerase and the Tm values of the primers and probes, and may be at least 25°C or higher. More preferably, it is 37°C or higher. In the PCR of 2, [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of primers to template single-stranded DNA, [3] the above using DNA polymerase Extension of the primer may be included, and [2] and [3] may be performed at the same temperature to form two steps. In order to perform rapid RT-PCR, the thermal cycler used for the RT-PCR reaction has a total elongation time of steps [2] and [3] of 60 seconds or less, preferably 45 seconds or less, More preferably, it is desirable to set the measurement program for 30 seconds or less. As used herein, the term "PCR extension time" refers to the set temperature of the thermal cycler.

The one-step RT-PCR solution added to the mixture contains reverse transcriptase and DNA polymerase. It is preferable to use Tth DNA polymerase, Taq DNA polymerase, etc., which are DNA polymerases that also have reverse transcriptase activity. More preferred is the use of two enzymes, the use of at least two enzymes, reverse transcriptase and DNA polymerase.

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

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

As used herein, a DNA polymerase mutant refers to, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% of the amino acid sequence of the wild-type DNA polymerase from which it is derived. % or more, preferably 99% or more sequence identity, and has the same activity as wild-type DNA polymerase for amplifying DNA and, if necessary, activity for converting RNA into cDNA. . Here, any method known in the art can be used to calculate the identity of amino acid sequences. For example, it can be calculated using a commercially available analysis tool or available through an electric communication line (Internet), for example, the homology algorithm BLAST (Basic local alignment search tool) http of the National Center for Biotechnology Information (NCBI) ://www. ncbi. nlm. nih. Amino acid sequence identity can be calculated by using default parameters in gov/BLAST/. Mutants that can be used in the present invention have one or several amino acid substitutions, deletions, insertions and/or additions (hereinafter collectively referred to as " It may be a polypeptide consisting of an amino acid sequence that has been mutated (also referred to as "mutated"), and have the same activities as wild-type DNA polymerase to convert RNA into cDNA and to amplify DNA. Here, one or several means, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, still more preferably 1 to 5, still more preferably 1 to 3. Possible, but not particularly limited.

The one-step RT-PCR reaction solution used in the present invention contains, in addition to reverse transcriptase and DNA polymerase, a buffer, appropriate salts such as magnesium salt or manganese salt, deoxynucleotide triphosphates, and viral RNA to be detected. It contains a pair of primers corresponding to the region to be detected, and if necessary, may contain additives.

Buffers used in the present invention include, but are not limited to, Tris, Tricine, Bis-Tricine, Bicine, and the like. It is adjusted to pH 6-9, more preferably pH 7-8, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like. Moreover, the concentration of the added buffer is 10 to 200 mM, preferably 20 to 150 mM. At this time, a salt solution is added to provide suitable ionic conditions for the reaction. Salt solutions include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, and the like.

As dNTPs used in the present invention, dATP, dCTP, dGTP and dTTP are each added at 0.1 to 0.5 mM, most generally about 0.2 mM. Precautions against cross-contamination may be taken by using dUTP instead of and/or as part of dTTP. Magnesium salts include magnesium chloride, magnesium sulfate and magnesium acetate, and manganese salts include manganese chloride, manganese sulfate and manganese acetate.

Furthermore, as additives contained in the one-step RT-PCR reaction solution, 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"), It preferably contains at least one selected from the group consisting of bovine serum albumin, glycerol, glycol, gelatin, and polar organic solvents.

Examples of the betaine-like quaternary ammonium salt include betaine (trimethylglycine), L-carnitine, etc. 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 possessed by betaine-like quaternary ammonium salts is a compound with stable positive and negative charges in the molecule, which exhibits surfactant-like properties and is thought to cause destabilization of virus structure. In addition, it is known to facilitate nucleic acid amplification of DNA polymerases. Preferably, the betaine-like quaternary ammonium salt concentration is 0.1M to 2M, more preferably 0.2M to 1.2M.

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

The gelatin contained in the one-step RT-PCR reaction solution is derived from the skin, bones, and tendons of animals such as bovines and pigs, or the scales and skins of fish, and is believed to contribute to the stabilization of PCR enzymes. The concentration used is preferably such that it does not interfere with fluorescence detection while stabilizing PCR amplification. It is preferably 1 to 5%, more preferably 1 to 2%. Although the origin of the gelatin is not particularly limited, gelatin derived from fish is preferable to gelatin derived from bovine or porcine in terms of lower jelly strength and better handling of the reaction solution.

Furthermore, it can be used in combination with substances known in the art to promote RT-PCR. Facilitators 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, dimethylsulfoxide (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-114, Tween 20, Nonidet P40, Briji 58, and the like. 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.

Furthermore, the one-step RT-PCR reaction solution may contain a polar organic solvent as an additive. Polarity refers to the electronic bias that exists within a molecule, and a molecule in which the centers of gravity of the positive and negative charges in the molecule do not coincide is called a polar molecule. Solvents composed of polar molecules are called polar solvents. Among polar solvents, the use of polar organic solvents composed of organic compounds can destabilize the higher-order structures of biomolecules such as nucleic acids and proteins. By utilizing this property, it is possible to weaken the hydrophobic bond of the viral structural protein and destabilize the capsid structure during the one-step RT-PCR reaction or in the pretreatment process. It is known that the capsid structure destabilization effect of polar organic solvents on viruses varies depending on the type of virus. It is considered that this is because the strength of hydrophobic binding and the like differs due to differences in the properties of capsid proteins and envelopes possessed by viruses.

Specific examples of the polar organic solvent include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphorictriamide, dimethylsulfoxide, acetone, acetonitrile, and ethanol. , methanol, 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Preferred are methanol, triethylamine, dimethylsulfoxide and acetone. A mixed solution containing two or more of these polar organic solvents may also be used. The lower limit concentration of the polar organic solvent as a capsid protein denaturing agent is not particularly limited as long as it is a concentration at which the capsid protein is denatured, although it depends on the types of the polar organic solvent and other additives. Also, since the capsid protein differs depending on the type of virus, the effective concentration of the polar solvent differs for each virus. % or more and 90% or less, more preferably 50% or more and 85% or less.

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

Said polar organic solvents are also commonly known as inhibitors of PCR. Therefore, among the above-mentioned polar organic solvents, by selecting a polar organic solvent with a small difference between the concentration required for protein denaturation and the allowable concentration to be carried into PCR, the polar organic solvent, the sample, and the one-step RT-PCR reaction solution are sequentially added. By doing so, the detection operation can be easily carried out in the same container from denaturation of the capsid protein to the one-step RT-PCR reaction without opening and closing the container during the process. An example of such a polar organic solvent is particularly preferably dimethylsulfoxide. For example, when 1 μL of dimethyl sulfoxide and 1 μL of sample are mixed and 48 μL of 1-step RT-PCR solution is added, the concentration of dimethyl sulfoxide brought into the reaction solution is 2%. 2% dimethyl sulfoxide is the allowable concentration to be brought into the RT-PCR solution.

In the method of the present invention, the one-step RT-PCR reaction solution in step (1) preferably further contains one or more primer pairs corresponding to the target region. The primer pair used in the present invention is, for example, a primer pair corresponding to the target region in the genomic RNA of the RNA virus when the detection target is an RNA virus contained in the sample, one primer pairing the other primer pair. Two pairs of primers are included that are complementary to each other in the DNA extension products of the primers. Another aspect is so-called multiplex PCR, in which two or more pairs of the above primers are included. In multiplex PCR, which specifically amplifies two or more target regions in one one-step RT-PCR reaction solution, a nucleic acid amplification reaction is performed with a plurality of primer pairs coexisting, so primer dimers and non-specific amplification occur. likely to occur. As shown in the results of the test examples described later, the present invention suppresses non-specific reactions even when such multiplex PCR that amplifies two or more target regions is performed by one-step RT-PCR. target nucleic acid can be amplified at high speed, enabling highly sensitive detection. As used herein, a "target region" is a nucleic acid sequence region of interest to be amplified by a primer pair, and can be, for example, a region intended for amplification in genomic RNA of viral RNA contained in a sample. As one preferred example, it may be possible to perform multiplex PCR using two or more regions in the genomic RNA of the viral RNA contained in the sample as two or more target regions. In the method of the present invention, the number of targets to be processed is not particularly limited, but may be two or more, for example, three or four. Although the upper limit of the number of targets is not particularly limited, it can be set to 10 or less, for example. Preferred is multiplex PCR that specifically amplifies two target regions in one one-step RT-PCR reaction solution.

Furthermore, degenerate primers may be included when the target nucleic acid consists of subtypes. When detecting a coronavirus (SARS-nCOV-2), which is a type of envelope RNA virus in the present invention, examples of primer pairs include "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases. Sequences described in (SEQ ID NOS: 1, 2, 4, 5), "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR PanelPrimers and Probes" released by the American Center for Disease Control and Prevention (SEQ ID NOS: 7, 8 , 10, 11, 13, 14), and can be preferably used in the present invention, but is not limited to these. In the primer sequences listed above, the nucleocapsid protein (N) region of SARS-nCOV-2 according to SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14 to detect In the detection of coronaviruses including SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame A gene such as an (ORF) region can be targeted for detection, but is not particularly limited to this. As for the concentration of the primers used, it is preferable that the concentration of the forward primer is 0.1 μM or more and 3 μM or less and the concentration of the reverse primer is 0.1 μM or more and 3 μM or less with respect to the entire RT-PCR reaction solution. . More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less.

Another aspect of the present invention is a detection method further comprising at least one labeled hybridization probe or double-stranded DNA-binding fluorescent compound. This allows the analysis of amplification products to be monitored by monitoring fluorescence signals instead of ordinary electrophoresis, thus reducing the analysis effort. Furthermore, it is possible to reduce the risk of contamination since there is no need to open the reaction vessel. It is also possible to distinguish virus subtypes by labeling each hybridization probe corresponding to a virus subtype with a different fluorescent dye.

Double-stranded DNA-binding fluorescent compounds include, for example, SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (registered trademark; Biotium). , LC Green (Idaho), LightCycler® 480 ResoLight (Roche Applied Science), and the like.

Hybridization probes used in the present invention include, for example, TaqMan hydrolysis probes (U.S. Pat. Nos. 5,210,015, 5,538,848, 5,487,972 , U.S. Pat. No. 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801), FRET hybridization probes (WO 97/46707, WO 97/46712) , International Publication No. 97/46714 pamphlet) and the like. The base sequence of the probe for detecting coronavirus coronavirus (SARS-nCOV-2), which is a type of enveloped RNA virus, is "2019-Novel Coronavirus (2019-nCoV) Real-time" announced by the Centers for Disease Control and Prevention. RT-pCR PanelPrimers and Probes" (SEQ ID NOS: 9, 12, 15) and the sequences described in the "Pathogen Detection Manual 2019-nCoV" published by the National Institute of Infectious Diseases (SEQ ID NOS: 3, 6), Although it can be suitably used in the present invention, it is not limited to this. The probe sequences described above detect the N region of SARS-nCOV-2. Furthermore, if the targeted nucleic acid consists of subtypes, it may contain degenerate sequences. In the detection of coronaviruses such as SARS-nCOV-2, genes such as the N region, E region, S region, RdRp region, and ORF region can be targeted for detection, but are not particularly limited to these. No. The concentration of the fluorescence-labeled probe is preferably 0.01 μM or more and 1.0 μM or less. It is more preferably 0.013 μM or more and 0.75 μM or less, and still more preferably 0.02 μM or more and 0.5 μM or less.

In the present invention, the method for determining whether non-specific nucleic acid amplification is suppressed is not limited, but the amplified product after RT-PCR is subjected to electrophoresis, and It can be confirmed by comparing with the band estimated from the number. In electrophoresis, it is possible to qualitatively determine the amount of non-specific nucleic acid amplification based on the intensity of the migration band. Another aspect is a detection method involving a double-stranded DNA binding fluorescent compound. By performing melting curve analysis in this method, amplification of the target nucleic acid and non-specific nucleic acid amplification can be distinguished from the difference in Tm values of the amplification products. By comparing the peak heights in the melting curves, it is possible to determine the amount of non-specific nucleic acid amplification. In addition, as still another form, it can be confirmed by a detection method using a hybridization probe (for example, TaqMan hydrolysis probe). In the TaqMan hydrolysis probe detection system, the fluorescence intensity increases according to the amount of amplified nucleic acid, and the PCR reaction cycle number at which the threshold fluorescence intensity is reached is called the Ct value. Increased non-specific nucleic acid amplification causes a decrease in incoming fluorescence intensity. Therefore, it is possible to determine the amount of non-specific nucleic acid amplification by comparing the reaching fluorescence intensity.

Another aspect of the invention is a composition for amplifying nucleic acids from template RNA in a sample by one-step RT-PCR with reduced non-specific nucleic acid amplification. In certain preferred embodiments, a composition for use in one-step RT-PCR characterized in that it contains an anionic polymer as described above and a reverse transcriptase and a DNA polymerase (or a DNA polymerase with reverse transcription activity) be. The present invention can also be provided in the form of a kit containing these compositions for use in one-step RT-PCR with suppressed non-specific nucleic acid amplification.

In one embodiment, the present invention can be a reagent containing an anionic polymer that is used to suppress non-specific amplification in nucleic acid amplification by one-step RT-PCR from a sample that has not undergone a purification process. In yet another embodiment, the present invention provides a one-step RT-PCR that specifically amplifies two or more target regions from a sample in one reaction solution. can be a reagent containing a reactive polymer. The method of use such as the type and amount of the anionic polymer used in these reagents, and other additives that can be used together may be the same as those described in detail in the nucleic acid amplification method.

Furthermore, the present invention relates to a kit for use in a nucleic acid amplification method by a one-step RT-PCR reaction, containing reagents as described above. For example, the kit of the present invention may include an anionic polymer and (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase having reverse transcription activity and other components that may optionally be used in the same container. or in separate containers, for example, can be packaged in one package and provided in a manner that includes information on how to use the kit. By using the kit of the present invention, it becomes possible to perform a nucleic acid amplification reaction in which non-specific reactions are suppressed, and it is possible to detect a target nucleic acid with high sensitivity.

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

Test example 1. Effects of anionic polymers on RT-PCR inhibition by saliva specimens (effects in multiplex PCR):
(1) Preparation of reaction solution The reaction solution with the composition shown below is used as a basic composition, and in 1-step RT-PCR, the reaction solution in the presence of a saliva sample (in the presence of unpurified biological contaminants) Inactivated SARS-nCOV-2 coronavirus RNA was detected. SARS-CoV-2 Detection Kit -N1 set- and -N2 set- (Toyobo) were used as detection reagents except for the pretreatment solution. In addition, the primers and probes for detection, which are attached to this reagent, are published by the American Centers for Disease Control and Prevention (CDC) "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective : 24 Jan 2020) and used at the concentrations indicated. The N1 probe was modified with Cy5 as a fluorescent label and BHQ3 (black hole quencher) as a quenching group, and the N2 probe was modified with ROX as a fluorescent label and BHQ2 as a quenching group.
RT-PCR reaction solution (40 μL)
Reaction solution: 30 μL
Enzyme solution: 5 μL
Primer/probe solution: 5 μL
(2) Addition of inactivated virus and saliva and pretreatment Pretreatment solution (10 μL)
10 copies/μL inactivated SARS-nCOV-2 (Zeptometrix): 1 μL
Saliva: 0 μL, 3 μL or 8 μL
Sodium polyvinyl sulfonate (PVSA): 1 μL (added at a concentration such that the final concentration in the reaction solution after addition of the 1-step RT-PCR reaction solution is 0% to 0.04%)
RNAse free water: adjusted to 10 µL 10 µL of the mixture was heat-treated at 95°C for 5 minutes in a thermal cycler.
(3) Addition of reaction solution 40 µL of the RT-PCR reaction solution prepared in (1) was added to 10 µL of the mixed solution after heat treatment in the previous step, and RT-PCR was performed in a 50 µL reaction system.
(4) RT-PCR Reaction Conditions Using BioRad CFX96WELL DEEP, a real-time PCR reaction was performed with the following temperature cycle.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 seconds (thermal denaturation)
95°C 1 sec - 50°C 3 sec - 55°C 10 sec 50 cycles (PCR - fluorescence reading)
(5) Results As for the measurement results, the reached fluorescence intensity was calculated using BioRad's CFX Manager or CFX Maestro software. The results are shown in FIG. 1 (with 3 μL of saliva added) and FIG. 2 (with 8 μL of saliva added). As a result, when saliva is added, the intensity of fluorescence reaching both N1 and N2 decreases under conditions in which PVSA is not present. It was suggested that

Test example 2. Effect of anionic polymers on sensitivity to RT-PCR inhibition by saliva samples (1) Preparation of reaction solution The reaction solution with the composition shown below is used as a basic composition, and 1-step RT-PCR is performed in the presence of saliva samples. Inactivated SARS-nCOV-2 viral RNA was detected in the reaction mixture (in the presence of unpurified biological contaminants). SARS-CoV-2 Detection Kit -N2 set- (Toyobo) was used as a detection reagent except for the pretreatment solution. In addition, the primers and probes for detection, which are attachments of this reagent, are published by the American Centers for Disease Control and Prevention (CDC) "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective : 24 Jan 2020) and used at the indicated concentrations. The N2 probe used was modified with ROX as a fluorescent label and BHQ2 as a quenching group.
RT-PCR reaction solution (40 μL)
Reaction solution: 30 μL
Enzyme solution: 5 μL
Primer/probe solution: 5 μL
(2) Addition of inactivated virus and saliva and pretreatment Pretreatment solution (10 μL)
10 copies/μL or 2.5 copies/μL Inactivated SARS-nCOV-2 (Zeptometrix): 1 μL
Saliva: 0 μL, 3 μL
Sodium polyvinyl sulfonate (PVSA): 1 μL (added at a concentration such that the final concentration in the reaction solution after addition of the 1-step RT-PCR reaction solution is 0.008%)
RNAse free water: adjusted to 10 µL 10 µL of the mixture was heat-treated at 95°C for 5 minutes in a thermal cycler.
(3) Addition of reaction solution 40 µL of the RT-PCR reaction solution prepared in (1) was added to 10 µL of the mixed solution after heat treatment in the previous step, and RT-PCR was performed in a 50 µL reaction system.
(4) RT-PCR Reaction Conditions Using BioRad CFX96WELL DEEP, a real-time PCR reaction was performed with the following temperature cycle.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 seconds (thermal denaturation)
95°C 1 sec - 50°C 3 sec - 55°C 10 sec 50 cycles (PCR - fluorescence reading)
(5) Results As for the measurement results, the Ct value and the reached fluorescence intensity were calculated with a threshold of 100 using BioRad's CFX Manager or CFX Maestro software. The results are shown in Table 1 below and FIG. As shown in these results, when saliva was added, the reaching fluorescence intensity decreased in the absence of PVSA, and the detection of 2.5 copies/μL could not be confirmed. On the other hand, in the presence of PVSA, the fluorescence intensity reached increased, and all detections up to 2.5 copies/μL were confirmed, indicating that the sensitivity was improved.

INDUSTRIAL APPLICABILITY The present invention is suitably used in molecular biological research, clinical examination, food hygiene management, and the like.

Claims (28)

A method for amplifying nucleic acids from a sample by one-step RT-PCR, comprising the steps of:
(1) preparing a one-step RT-PCR reaction solution comprising a sample, an anionic polymer, and (i) a reverse transcriptase and a DNA polymerase or (ii) a DNA polymerase with reverse transcription activity; and (2) After sealing the reaction vessel, performing a one-step RT-PCR reaction;
and wherein the concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more. 2. The nucleic acid amplification method according to claim 1, wherein the sample used in step (1) is a biological sample, an environmental sample, or a cell-derived sample that has not undergone a purification step. The sample used in the step (1) is saliva, sputum, pharyngeal swab, nasal swab, gargle, feces, lung aspirate, cerebrospinal fluid, tears, cultured cells, culture supernatant, swab test sample, 3. The nucleic acid amplification method according to claim 1, wherein the sample is at least one selected from the group consisting of soil samples and sewage samples. The sample used in step (1) is suspended in at least one selected from the group consisting of water, physiological saline, buffer, and sputazyme enzyme solution, or a centrifugal supernatant thereof, or 4. The nucleic acid amplification method according to any one of claims 1 to 3, which is a concentrate. 5. The nucleic acid amplification method according to any one of claims 1 to 4, wherein the sample used in step (1) is a sample that may contain an RNA virus. 6. The nucleic acid amplification method according to any one of claims 1 to 5, wherein two or more target regions are specifically amplified in one one-step RT-PCR reaction solution. 7. The nucleic acid amplification method according to claim 6, wherein the two or more target regions are target regions in genomic RNA of an RNA virus that can be contained in the sample. The nucleic acid amplification method according to any one of claims 5 to 7, wherein the RNA virus is an enveloped RNA virus. The enveloped RNA viruses consist of coronavirus family viruses, flaviviridae viruses; togaviridae viruses; orthomyxoviridae viruses; rhabdoviridae viruses; bunyaviridae viruses; The nucleic acid amplification method according to claim 8, wherein the nucleic acid is at least one selected from the group. The enveloped RNA virus is at least one selected from the group consisting of SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2 coronavirus. 9. The nucleic acid amplification method according to 8 or 9. The nucleic acid amplification method according to any one of claims 5 to 7, wherein the RNA virus is a non-enveloped RNA virus. 11. The non-enveloped RNA virus is at least one selected from the group consisting of Caliciviridae virus, Astroviridae virus; Picornaviridae virus; Hepeviridae virus; and Reoviridae virus. The nucleic acid amplification method according to . The anionic polymer is a polymer obtained by polymerizing a monomer having at least one anionic functional group selected from the group consisting of a sulfonic acid group, a carboxyl group, a phosphoric acid group, a sulfuric acid group, and a phosphonic acid group. The nucleic acid amplification method according to any one of claims 1 to 12. The nucleic acid amplification method according to any one of claims 1 to 13, wherein the anionic polymer has an average molecular weight of 1,000 to 5,000,000. 15. The nucleic acid amplification method according to any one of claims 1 to 14, wherein the DNA polymerase is at least one selected from the group consisting of Taq, Tth and variants thereof. Claims 1 to 5, wherein the origin of the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV), and variants thereof. 16. The nucleic acid amplification method according to any one of 15. The nucleic acid amplification method according to any one of claims 1 to 16, wherein the one-step RT-PCR reaction solution in step (1) further contains one or more primer pairs corresponding to the target region. The nucleic acid amplification method according to any one of claims 1 to 17, wherein the one-step RT-PCR reaction solution in step (1) further contains a hybridization probe corresponding to the target region. The one-step RT-PCR reaction solution in the step (1) contains a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine 19. The nucleic acid amplification method according to any one of claims 1 to 18, further comprising at least one selected from the group consisting of serum albumin, glycerol, glycol, gelatin, and polar organic solvents. 20. The nucleic acid amplification method according to claim 19, wherein the betaine-like quaternary ammonium salt is betaine or L-carnitine. The nucleic acid amplification method according to any one of claims 1 to 20, wherein the concentration of the anionic polymer in the one-step RT-PCR reaction solution in step (1) is 0.0025 to 0.05%. A method for suppressing non-specific amplification, wherein an anionic polymer is allowed to coexist in a one-step RT-PCR reaction solution in nucleic acid amplification by one-step RT-PCR from a sample that has not undergone a purification step. 23. The method for suppressing non-specific amplification according to claim 22, wherein an anionic polymer is allowed to coexist in an amount of 0.001% or more in the one-step RT-PCR reaction solution. A method for nucleic acid amplification by one-step RT-PCR for specifically amplifying two or more target regions from a sample in one reaction solution, characterized in that an anionic polymer is allowed to coexist in the reaction solution. Method. A reagent containing an anionic polymer, which is used to suppress non-specific amplification in nucleic acid amplification by one-step RT-PCR from a sample that has not undergone a purification step. 26. The reagent according to claim 25, which is adjusted so that the concentration of the anionic polymer in the one-step RT-PCR reaction solution is 0.001% or more. A reagent for use in the method for nucleic acid amplification by one-step RT-PCR reaction according to any one of claims 1 to 24, which contains an anionic polymer. A kit for use in a nucleic acid amplification method by a one-step RT-PCR reaction, containing the reagent according to any one of claims 25 to 27.

Images