JP2022153338A - Improvement method of preservation stability of nucleic acid amplification composition - Google Patents

Abstract

To provide a nucleic acid amplification composition capable of improving preservation stability.SOLUTION: There is provided a nucleic acid amplification composition including: (I) a DNA polymerase; (II) nucleotide which is, oligonucleotide and/or mononucleotide; (III) a divalent or greater-valent metal ion; (IV) a buffer agent; and (V) an antioxidant. The antioxidant is selected from a group formed of: ascorbate; tocopherol; tocotrienol; carotene; xanthophyll; flavonoid; polyphenol; riboflavin; thiol; gallic acid ester; sulfurous acids; alkyl phenol; melatonin; urobilinogen; uric acid; bilirubin; nordihydroguaiaretic acid; coumarin; catalase; ferritin; albumin; thioredoxin; melanoidin; sulfur dioxide; trihydroxy-butyrophenone.SELECTED DRAWING: None

Description

The present invention relates to a method for improving storage stability of a composition for nucleic acid amplification. More specifically, a method for improving the storage stability of a composition for nucleic acid amplification by adding an antioxidant, a composition for nucleic acid amplification with improved storage stability thereby, and storage stability as described above The present invention relates to a method for detecting a nucleic acid by performing a real-time polymerase chain reaction or a real-time reverse transcription polymerase chain reaction using a nucleic acid amplification composition having an improved nucleic acid amplification.

Nucleic acid amplification is a technology that amplifies a few copies of a 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 consists of (1) 2-step RT-PCR in which RT and PCR are performed discontinuously, (2) RT and PCR in succession using a single enzyme 1-step RT -PCR, and (3) a two-enzyme one-step RT-PCR in which RT and PCR are successively performed using two enzymes, reverse transcriptase and DNA polymerase.

PCR and RT-PCR are widely used for genetic testing and pathogenic microorganism testing. A representative example of virus testing is norovirus, which is one of pathogenic RNA viruses. Norovirus is a single-stranded RNA virus that causes acute gastroenteritis. Since it is highly contagious and causes mass food poisoning and mass infections, it is a virus of great public health concern. No tissue culture method has been established for norovirus pathogen testing, and methods for detecting viral genes using electron microscopy, immunological antigen detection by ELISA, or nucleic acid amplification technology have been developed. Of these, in Japan, the RT-PCR method based on the notification of the Safety Division of the Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare (No. there is

In addition, a test method using nucleic acid amplification technology has been established for the SARS-CoV-2 mutant coronavirus, which was confirmed to occur in Wuhan City, Hubei Province, China in 2019 and has spread worldwide. Patent Document 1, Non-Patent Document 2), a huge number of tests are being conducted worldwide. In addition, methods using nucleic acid amplification technology have been implemented in the inspection of various pathogenic microorganisms (for example, RNA viruses, DNA viruses, bacteria, fungi, etc.).

Pathogenic microorganism testing is required to be able to efficiently test multiple specimens in a short time, in order to prevent the spread of infection at an early stage. For example, in the case of a test to detect RNA viruses such as norovirus and the mutant coronavirus SARS-CoV-2, a method that simplifies the work by omitting the laborious and time-consuming task of purifying RNA from specimens. have been developed (for example, Patent Document 1, Patent Document 2, Non-Patent Document 3).

Commercially available PCR reaction reagents are often divided into a plurality of parts, and when performing a test, it is necessary to mix each part and prepare a PCR reaction solution. When conducting tests at high frequencies, the work load for preparing PCR reaction mixtures is large. Therefore, attempts have been made to reduce the work load by preparing multiple tests at once. However, the prepared PCR reaction solution cannot maintain its stability for a long period of time, resulting in a problem of decreased sensitivity. For example, in a method for detecting an amplification product using a fluorescent compound, it is confirmed as a decrease or disappearance of the obtained fluorescence intensity. A decrease in the sensitivity of the reagent makes it impossible to carry out the test correctly, which may lead to false negatives. In addition, additional work is required in the case of retesting, resulting in loss of time and money.

Therefore, there is a demand for the development of a method for improving the storage stability of a nucleic acid amplification composition such as a PCR reaction solution while maintaining excellent workability.

WO2019/017452 Pamphlet WO2019/212023 pamphlet

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-time RT-PCR) Journal of Food Microbiology, Vol. 35, 2018, pp. 193-198 National Institute of Infectious Diseases website “Pathogen Detection Manual 2019-nCoV” (https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200217.pdf)

An object of the present invention is to provide a technique that has excellent workability, high storage stability as a composition for nucleic acid amplification, and can detect the presence or absence of a target nucleic acid with sufficient sensitivity even after long-term storage.

In view of the above circumstances, the present inventors conducted intensive research and found that the coexistence of an antioxidant with a nucleic acid amplification composition such as a PCR reaction solution can maintain the storage stability of the composition, To detect target nucleic acids (for example, target nucleic acids derived from pathogenic microorganisms such as viruses and bacteria) with sufficient sensitivity even when the composition is subjected to nucleic acid amplification reactions such as PCR reactions or RT-PCR reactions after storage. found that it is possible.

In general, the PCR reaction solution contains various components such as enzymes, nucleotides such as oligonucleotides, metal ions, fluorescent compounds for detection, buffer solutions, and additives required for the reaction. has been prepared. This is because storage of the PCR reaction solution has been thought to cause a significant decrease in detection sensitivity due to factors such as deterioration and decomposition of components in the reaction solution, and digestion of components due to unexpected reactions by enzymes. be. However, quite unexpectedly, the use of antioxidants such as ascorbic acids, tocopherols, sulfites, alkylphenols, etc. improves the storage stability of PCR reaction solutions due to their high antioxidant capacity. The present inventors have found that even after storage for a certain period of time, the decrease in detection sensitivity can be suppressed to a high degree, making it possible to detect the target nucleic acid using a fluorescent compound or the like. As a result, it is possible to prepare and store the PCR reaction solution collectively in advance, and it is possible to greatly omit the work of preparing the reaction solution, which is particularly burdensome when testing a large number of samples at high frequency. . As a result, the present inventors have found that efficient genetic testing and pathogenic microorganism testing can be performed using nucleic acid amplification reactions, and have completed the present invention.

The representative invention of the present application is as follows.
[Item 1] (I) a DNA polymerase, (II) at least one nucleotide selected from the group consisting of oligonucleotides and mononucleotides, (III) a metal ion having a valence of 2 or more, (IV) a buffer, and (V) ) A composition for nucleic acid amplification containing an antioxidant.
[Item 2] The composition for nucleic acid amplification according to Item 1, comprising (II) a probe fluorescently labeled as a nucleotide.
[Item 3] The composition for nucleic acid amplification according to item 1 or 2, further comprising (VI) an anti-DNA polymerase antibody.
[Item 4] (I) The composition for nucleic acid amplification according to any one of Items 1 to 3, wherein the DNA polymerase is a DNA polymerase having reverse transcription activity.
[Item 5] (I) The composition for nucleic acid amplification according to any one of Items 1 to 4, wherein the DNA polymerase belongs to Family A.
[Item 6] The composition for nucleic acid amplification according to any one of items 1 to 5, further comprising (VII) a reverse transcriptase.
[Item 7] (VII) The reverse transcriptase is selected from the group consisting of Moloney murine leukemia virus (MMLV)-derived reverse transcriptase, avian myeloblastosis virus (AMV)-derived reverse transcriptase, and variants thereof. 7. The composition for nucleic acid amplification according to Item 6, which is at least one of
[Item 8] The composition for nucleic acid amplification according to any one of Items 2 to 7, wherein the fluorescently labeled probe is a TaqMan probe.
[Item 9] The composition for nucleic acid amplification according to any one of items 1 to 8, wherein the concentration of (V) antioxidant is 0.001 mM or more.
[Item 10] (V) Antioxidants such as ascorbic acids, tocopherols, tocotrienols, carotenes, xanthophylls, flavonoids, polyphenols, riboflavins, thiols, gallates, sulfites, alkylphenols, At least one antioxidant selected from the group consisting of melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sulfur dioxide, and trihydroxybutyrophenone (THBP) Item 10. The composition for nucleic acid amplification according to any one of Items 1 to 9, comprising
[Item 11] (V) Antioxidants such as ascorbic acid, erythorbic acid, tocopherol, tocopherol acetate, tocopherol succinate, tocotrienol, tocotrienol acetate, tocotrienol succinate, retinol, α-carotene, β-carotene, lycopene, lutein, zeaxanthin, Canthaxanthin, astaxanthin, rubixanthin, β-cryptoxanthin, capsanthin, fucoxanthin, myxoxanthin, catechin, anthocyanin, tannin, rutin, isoflavone, isoflavane, nobiletin, chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, oleocanthal, Oleuropein, resveratrol, riboflavin, riboflavin phosphate, lutathione, cysteine, melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sodium sulfite, potassium sulfite, pyrro At least one antioxidant selected from the group consisting of potassium sulfite, propyl gallate, octyl gallate, dodecyl gallate, dibutylhydroxytoluene, butylhydroxyanisole, butylhydroquinone, sulfur dioxide, trihydroxybutyrophenone, and salts thereof Item 11. The composition for nucleic acid amplification according to any one of Items 1 to 10, comprising
[Item 12] The composition for nucleic acid amplification according to any one of Items 1 to 11, wherein (III) the divalent or higher metal ion comprises at least one selected from the group consisting of magnesium ion and manganese ion.
[Item 13] The composition for nucleic acid amplification according to any one of items 1 to 12, which is a premix type reagent for nucleic acid amplification.
[Item 14] The composition for nucleic acid amplification according to any one of items 1 to 13, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 4°C for 4 to 14 days.
[Item 15] The composition for nucleic acid amplification according to any one of items 1 to 14, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 25°C for 4 to 7 days.
[Item 16] The composition for nucleic acid amplification according to any one of Items 1 to 15, wherein the composition for nucleic acid amplification according to any one of Items 1 to 15 suppresses a change in color tone as measured by any absorbance in the 435 to 490 nm region when left at 25°C for 4 days. thing.
[Claim 17] A nucleic acid comprising (I) a DNA polymerase, (II) at least one nucleotide selected from the group consisting of oligonucleotides and mononucleotides, (III) a metal ion having a valence of 2 or more, and (IV) a buffer A method for improving the storage stability of a composition for nucleic acid amplification by allowing the composition for amplification to coexist with (V) an antioxidant.
[Item 18] The method according to Item 17, wherein the nucleic acid amplification composition contains (II) a probe fluorescently labeled as a nucleotide.
[Item 19] The method according to Item 17 or 18, wherein the composition for nucleic acid amplification further comprises (VI) an anti-DNA polymerase antibody.
[Item 20] (I) The method according to any one of Items 17 to 19, wherein the DNA polymerase is a DNA polymerase having reverse transcription activity.
[Item 21] (I) The method according to any one of Items 17 to 20, wherein the DNA polymerase belongs to Family A.
[Item 22] The method according to any one of Items 17 to 21, wherein the composition for nucleic acid amplification further comprises (VII) a reverse transcriptase.
[Claim 23] (VII) The reverse transcriptase is selected from the group consisting of reverse transcriptase derived from Moloney murine leukemia virus (MMLV), reverse transcriptase derived from avian myeloblastosis virus (AMV), and variants thereof. Item 23. The method according to Item 22, wherein at least one of the
[Item 24] The method according to any one of Items 18 to 23, wherein the fluorescently labeled probe is a TaqMan probe.
[Item 25] The method according to any one of Items 17 to 24, wherein (V) an antioxidant is allowed to coexist at a concentration of 0.001 mM or more in the nucleic acid amplification composition.
[Item 26] (V) Antioxidants such as ascorbic acids, tocopherols, tocotrienols, carotenes, xanthophylls, flavonoids, polyphenols, riboflavins, thiols, gallates, sulfites, alkylphenols, At least one antioxidant selected from the group consisting of melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sulfur dioxide, and trihydroxybutyrophenone (THBP) Item 26. The method according to any one of Items 17 to 25, wherein
[Item 27] (V) Antioxidants such as ascorbic acid, erythorbic acid, tocopherol, tocopherol acetate, tocopherol succinate, tocotrienol, tocotrienol acetate, tocotrienol succinate, retinol, α-carotene, β-carotene, lycopene, lutein, zeaxanthin, Canthaxanthin, astaxanthin, rubixanthin, β-cryptoxanthin, capsanthin, fucoxanthin, myxoxanthin, catechin, anthocyanin, tannin, rutin, isoflavone, isoflavane, nobiletin, chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, oleocanthal, Oleuropein, resveratrol, riboflavin, riboflavin phosphate, lutathione, cysteine, melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sodium sulfite, potassium sulfite, pyrro At least one antioxidant selected from the group consisting of potassium sulfite, propyl gallate, octyl gallate, dodecyl gallate, dibutylhydroxytoluene, butylhydroxyanisole, butylhydroquinone, sulfur dioxide, trihydroxybutyrophenone, and salts thereof Item 27. The method according to any one of items 17 to 26, wherein
[Item 28] The method according to any one of Items 17 to 27, wherein the composition for nucleic acid amplification contains (III) at least one selected from the group consisting of magnesium ions and manganese ions as the divalent or higher metal ion. .
[Item 29] The method according to any one of Items 17 to 28, wherein the composition for nucleic acid amplification is a premix type reagent.
[Item 30] The method according to any one of Items 17 to 29, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 4°C for 4 to 14 days.
[Item 31] The method according to any one of Items 17 to 30, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 25°C for 4 to 7 days.
[Item 32] The method according to any one of Items 17 to 31, wherein a change in color tone as measured by any absorbance within the 435 to 490 nm region is suppressed when left at 25°C for 4 days.

According to the present invention, the storage stability of nucleic acid amplification compositions such as PCR reaction solutions (including RT-PCR reaction solutions) is improved, and detection sensitivity is maintained. No need to prepare for errands. Improving the storage stability of the PCR reaction solution prevents the occurrence of false negatives and failure of the test due to the decrease in detection sensitivity, enables accurate testing, and reduces work such as retesting. In addition, by omitting the preparation work of the composition for nucleic acid amplification, which frequently occurs when handling a large number of specimens, the efficiency of genetic testing and pathogenic microorganism testing work is further improved. Improving the efficiency of pathogenic microorganism testing operations can increase the number of test subjects who are infected but show no symptoms, and can greatly contribute to preventing the spread of infectious diseases.

FIG. 3 shows the results of evaluating the storage stability at 25° C. when an antioxidant was added to the 1-enzyme system 1-step RT-PCR reaction solution. FIG. 2 shows the results of evaluating the storage stability at 4° C. when an antioxidant was added to the 1-enzyme system 1-step RT-PCR reaction solution. FIG. 2 shows the results of evaluating storage stability at 4° C. when an antioxidant was added to a two-enzyme system one-step RT-PCR reaction solution. FIG. 4 shows the results of using an RT-PCR reaction solution containing an antioxidant after storage at 4° C. for virus detection. FIG. 3 shows the results of evaluating the storage stability at 4° C. (detection of the N1 region) in the case where an antioxidant was added to a multiplex RT-PCR reaction solution of a two-enzyme system in one step. FIG. 2 shows the results of evaluating the storage stability at 4° C. (detection of the N2 region) in the case of adding an antioxidant to a two-enzyme system, one-step multiplex RT-PCR reaction solution. FIG. 2 shows the results of evaluation of storage stability at 4° C. (SARS-CoV-2 detection) when an antioxidant was added to a 2-enzyme system 1-step multiplex RT-PCR reaction solution. FIG. 2 is a diagram showing the results of evaluating the storage stability at 4° C. (influenza A detection) when an antioxidant was added to a 2-enzyme system 1-step multiplex RT-PCR reaction solution. Results of evaluating the storage stability at 4 ° C when sodium sulfite was added to the 2-enzyme system 1-step multiplex RT-PCR reaction solution (SARS-CoV-2 (SC2), influenza A (FluA) and influenza FIG. 10 is a diagram showing detection of type B (FluB).

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

In one aspect of the present invention, for example, (I) a DNA polymerase, (II) nucleotides such as oligonucleotides and/or mononucleotides, (III) divalent or higher metal ions, and (IV) a buffer, together with (V ) A composition for nucleic acid amplification characterized in that an antioxidant is coexistent.
The present invention can improve the storage stability of the nucleic acid amplification composition containing the components (I) to (IV) by coexisting (V) an antioxidant; Since the oxidizing agent does not interfere with the nucleic acid amplification reaction, fluorescent compounds detect the nucleic acids (DNA, RNA) of pathogenic microorganisms (including viruses, bacteria, fungi, etc.) in samples with sufficient sensitivity even when stored for a certain period of time. Based on the finding that it can be detected.

Antioxidant (also referred to as antioxidant) used in the present invention is a general term for substances having a function of suppressing the oxidation reaction of coexisting substances. In particular, in the present invention, the oxidation reaction of constituents contained in a nucleic acid amplification composition such as a PCR reaction solution is suppressed. The antioxidant used in the present invention may be a substance derived from natural ingredients or a non-naturally derived substance. Moreover, the antioxidant may be a water-soluble antioxidant or a fat-soluble antioxidant. When using a fat-soluble antioxidant, for example, it may be dissolved in advance in a polar organic solvent such as dimethylsulfoxide (DMSO) and added to the composition for nucleic acid amplification. In the present invention, the antioxidants can be used singly or in combination of two or more, regardless of whether they are naturally derived or non-naturally derived, and whether they are water-soluble antioxidants or fat-soluble antioxidants.

Examples of antioxidants derived from natural ingredients include ascorbic acids such as ascorbic acid (vitamin C) and erythorbic acid (isoascorbic acid); tocopherols such as tocopherol, tocopherol acetate and tocopherol succinate; tocotrienols such as tocotrienol succinate; carotenes such as retinol (vitamin A), α-carotene, β-carotene, and lycopene; xanthophylls such as; catechin, anthocyanin, tannin, rutin, isoflavone, isoflavane, flavonoids such as nobiletin; polyphenols such as chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, oleocanthal, oleuropein, resveratrol; riboflavin, riboflavins such as riboflavin phosphate; thiols such as glutathione and cysteine; melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, derivatives thereof, and their derivatives (eg, potassium salt, sodium salt, hydrate salt) and the like, but are not particularly limited. Ascorbic acids and tocopherols are preferable as antioxidants derived from natural ingredients, and ascorbic acid, erythorbic acid, and tocopherol acetate (DL-α-tocopherol acetate, etc.), from the viewpoint that the effects of the present invention can be more reliably obtained. , and salts thereof are more preferred.

Examples of non-naturally derived antioxidants include sulfites such as sodium sulfite, potassium sulfite, and potassium pyrosulfite; gallic acid esters such as propyl gallate, octyl gallate, and dodecyl gallate; and dibutylhydroxytoluene (BHT). , butylhydroxyanisole (BHA), butylhydroquinone (BHQ) and other alkylphenols; sulfur dioxide, trihydroxybutyrophenone (THBP), derivatives thereof, and salts thereof (e.g., potassium salt, sodium salt, hydrate salt ) and the like, but are not particularly limited. From the viewpoint that the effects of the present invention can be more reliably obtained, the non-natural component-derived antioxidants are preferably sulfites, gallic acid esters, and alkylphenols. Anisole (BHA), butylhydroquinone (BHQ), and salts thereof are more preferred.

In the present invention, the antioxidant concentration (final concentration during storage) contained in the nucleic acid amplification composition is not particularly limited, but is usually 50 mM or less, preferably 50 mM or less, relative to the entire nucleic acid amplification composition (eg, PCR reaction reagent). is 20 mM or less, more preferably 10 mM or less, more preferably 7 mM or less, and even more preferably 6 mM or less. Although the lower limit is not particularly limited, it can be, for example, 0.001 mM or more relative to the entire nucleic acid amplification composition (eg, PCR reaction reagent).

In certain embodiments, the final concentration of the antioxidant in the reaction solution during nucleic acid amplification reaction such as PCR reaction or RT-PCR reaction can be, for example, 0.001 to 50 mM, and can be 0.01 to 20 mM. is preferably 0.1 to 10 mM, and even more preferably 0.5 to 6 mM. Even when the antioxidant is present at such a final concentration in the reaction solution, it is possible to carry out the nucleic acid amplification reaction with sufficient sensitivity.

The composition for nucleic acid amplification of the present invention may contain a DNA polymerase. The DNA polymerase is a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or a DNA polymerase that can use both DNA and RNA as a template (i.e., a DNA polymerase with reverse transcriptase activity). good too. Further, the DNA polymerase may be a DNA polymerase belonging to any family, but a DNA polymerase belonging to Family A is preferable from the viewpoint that the effects of the present invention can be more reliably obtained. In the present invention, any DNA polymerase known in the art can be appropriately selected and used according to the purpose of nucleic acid amplification.

In a preferred embodiment, the DNA polymerase used in the present invention is, for example, a contaminant-tolerant DNA polymerase. Contaminant resistance refers to the property of a DNA polymerase having high enzymatic activity sufficient for nucleic acid amplification reactions, for example, even in the presence of PCR inhibitors. The contaminant-resistant DNA polymerase is not particularly limited, and examples thereof include Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEP VENT, HawkZ05, and mutations thereof. Examples include, but are not limited to, bodies. Preference is given to using Tth (SEQ ID NO: 24) and HawkZ05 (SEQ ID NO: 25) or variants thereof. Particularly preferred is the use of Tth or variants thereof. Even DNA polymerases such as Taq, which normally do not have contamination resistance, can be used if they are mutants that have contamination resistance due to amino acid mutation. The total amount of the contaminant-resistant DNA polymerase in the nucleic acid amplification composition of the present invention may be, for example, at least 4.2 ng/μL or more, preferably 5.0 ng/μL or more, and 5.8 ng. /μL or more is more preferable. Among them, it is preferably 8.3 ng/μL or more. The upper limit of the total amount of the contaminant-resistant DNA polymerase is not particularly limited, but as an example, it can be 20 ng/μL or less, and even if it is 16.7 ng/μL or less, the effect of the present invention can be sufficiently obtained. can be done. The amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be estimated from the Safety Data Sheet (SDS). When protein such as BSA is included, it is desirable to calculate by the latter method. In order to enhance the effect of suppressing non-specific reactions, enzymatic activity of DNA polymerase is suppressed until the start of PCR reaction by using it in combination with an anti-DNA polymerase antibody or introducing a thermolabile blocking group into DNA polymerase by chemical modification. , preferably applicable to hot-start PCR.

When the nucleic acid amplification composition of the present invention is used for nucleic acid amplification including a step of reverse transcription reaction, the composition for nucleic acid amplification of the present invention contains a DNA polymerase and, if necessary, a reverse transcriptase. is preferred. The origin of the reverse transcriptase (RT) that can be used in the present invention is not particularly limited as long as it is an enzyme that has the activity of converting RNA into DNA. Avian Myeloblastosis Virus), HIV-RT, RAV2-RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and variants thereof. Particularly preferred examples include reverse transcriptase from Moloney murine leukemia virus (MMLV-RT), reverse transcriptase from avian myeloblastosis virus (AMV-RT), or variants thereof.

The reverse transcriptase may be a DNA polymerase that also has reverse transcriptase activity. A DNA polymerase with reverse transcription activity is a DNA polymerase that has both the ability to convert RNA into cDNA and the ability to amplify DNA. A DNA polymerase having reverse transcription activity preferably has contaminant resistance and/or heat resistance. The term "heat resistance" means that the enzymatic activity does not decrease by more than half even after heat treatment at 70°C for 1 minute or more. Although the origin is not particularly limited, Taq, Tth, Bst, Bca, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and variants thereof can be mentioned. As DNA polymerases having reverse transcription activity, thermus aquaticus-derived DNA polymerase (Taq), Thermus thermophilus HB8-derived DNA polymerase (Tth), Thermus sp Z05-derived DNA polymerase (Z05), and Thermotoga maritima-derived DNA polymerase have been used. (Tma), Bacillus caldotenax-derived DNA polymerase (Bca), Bacillus stearothermophilus-derived DNA polymerase (Bst), etc., and even mutants that have not lost their reverse transcription activity and thermostable DNA polymerase activity. good. In addition, mutants of DNA polymerase (KOD) derived from Thermococcus kodakaraensis and having reverse transcription activity are known (e.g., RTX: reverse transcription xenopolymerase), and the present invention includes such a reverse transcriptase activity A heat-stable DNA polymerase having both can also be used. Particularly preferred are DNA polymerases having reverse transcription activity selected from the group consisting of Tth (SEQ ID NO: 24), Z05 (SEQ ID NO: 25) and variants thereof.

As used herein, a mutant of DNA polymerase or reverse transcriptase refers to, for example, 85% or more, preferably 90% or more, more preferably 90% or more of the amino acid sequence of the wild-type DNA polymerase or reverse transcriptase from which it is derived. have a sequence identity of 95% or more, more preferably 98% or more, and most preferably 99% or more, and their corresponding wild-type enzyme activity (DNA polymerase activity or reverse transcriptase activity) is substantially It means something that is not physically damaged. In the case of a DNA polymerase that also has reverse transcription activity, it means that the activity of converting RNA into cDNA and the activity of amplifying DNA are not substantially impaired. 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/. In addition, mutants that can be used in the present invention have one or several amino acid substitutions, deletions, insertions and/or additions in the amino acid sequence of the wild-type DNA polymerase or reverse transcriptase from which they are derived (hereinafter referred to as are collectively referred to as "mutation"), and the activity of their corresponding wild-type enzymes (DNA polymerase activity or reverse transcriptase activity) is not substantially impaired. There may be. Here, one or several may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, still more preferably 1 to 5, but is not particularly limited.

The composition for nucleic acid amplification of the present invention may contain nucleotides. Nucleotides may be mononucleotides or oligonucleotides. Mononucleotides include nucleotides (including nucleotide monophosphates, nucleotide diphosphates, nucleotide triphosphates, etc.), deoxynucleotides (including deoxynucleotide monophosphates, deoxynucleotide diphosphates, deoxynucleotide triphosphates, etc.) , nucleosides, deoxynucleosides and the like. Oligonucleotides can be polynucleotides of any length, and can generally be oligonucleotides of about 2 to 40 bases in length, preferably about 2 to 25 bases in length. Nucleotides that can be contained in the nucleic acid amplification composition of the present invention can be, for example, deoxynucleotide triphosphates (dNTPs), primers, hybridization probes (including fluorescently labeled probes), etc., but are limited to these. not.

In one embodiment, the composition for nucleic acid amplification of the present invention may contain deoxynucleotide triphosphates (dNTPs) as nucleotides. The dNTPs used in the present invention may be a mixture containing dATP, dCTP, dGTP, and dTTP each at about 0.1 to 0.5 mM, generally about 0.2 mM. By using dUTP instead of and/or as part of dTTP, it is also possible to take precautions against cross-contamination using Uracil-N-glycosylase (UNG).

The composition for nucleic acid amplification of the present invention may contain, as nucleotides, nucleic acid primers designed to amplify a predetermined region of a target nucleic acid. When the composition for nucleic acid amplification of the present invention is used for PCR reaction, primers can be used as a primer pair. Primer pairs for use in the present invention include two pairs of primers in which one primer is mutually complementary to the DNA extension product of the other primer. Although not particularly limited, the present invention may include two or more pairs of the above primers. Furthermore, degenerate primers may be included when the target nucleic acid consists of subtypes.

In one embodiment, for example, when detecting norovirus with the nucleic acid amplification composition of the present invention, as an example of a primer pair for detecting norovirus, the notification of the Safety Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare (Food Safety Inspection 1105001 No.), but not limited to the primers (SEQ ID NOs: 1 to 5) described in With the primers, Norovirus G1 type is detected by SEQ ID NOs: 1 and 2, and Norovirus G2 type is detected by SEQ ID NOs: 3-5. The concentration of the primer to be detected is not particularly limited, but 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 with respect to the entire RT-PCR reaction solution. The following are preferred. 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.

In another embodiment, when detecting a novel coronavirus (SARS-CoV-2) with the nucleic acid amplification composition of the present invention, examples of primer pairs include "Pathogen Detection" published by the National Institute of Infectious Diseases. Manual 2019-nCoV" sequence (SEQ ID NO: 9, 10, 12, 13), "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers and Probes" published by the American Center for Disease Control and Prevention (SEQ ID NOS: 15, 16, 18, 19, 21, 22), which can also be preferably used in the present invention, but are not limited thereto. Said primer sequences detect the nucleocapsid protein (N) region of SARS-CoV-2 according to SEQ ID NOs: 9 and 10, 12 and 13, 15 and 16, 18 and 19, 21 and 22. In the detection of coronaviruses including SARS-CoV-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.

In another embodiment, when detecting an influenza virus with the nucleic acid amplification composition of the present invention, examples of primer pairs include "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes” (SEQ ID NOS: 26-29, 31, 32), etc., which can be preferably used in the present invention, but are not limited thereto. In the primer sequences described above, SEQ ID NOs: 26 to 29 detect influenza A, and SEQ ID NOs: 31 and 32 detect influenza B, but the primer sequences are not particularly limited to these. 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.

The composition for nucleic acid amplification of the present invention may contain, as nucleotides, a nucleic acid probe designed to detect amplification of a target nucleic acid (for example, a probe consisting of a fluorescently labeled oligonucleotide). When the composition for nucleic acid amplification of the present invention is used for real-time PCR reaction or real-time RT-PCR reaction, for example, by using a hybridization probe labeled with a fluorescent compound, fluorescence Signal monitoring can monitor for amplification of the target nucleic acid, and the risk of contamination is reduced because the reaction vessel does not need to be opened to detect nucleic acid amplification. It is also possible to separately identify each target nucleic acid, for example, by labeling each hybridization probe corresponding to the type or subtype of viruses and microorganisms to be detected with different fluorescent dyes.

A composition for nucleic acid amplification containing the fluorescently labeled probe as described above is allowed to coexist with other components (for example, various components such as enzymes, metal ions, buffer solutions, additives, etc.) necessary for nucleic acid amplification reaction. It is known that when stored in this state for a long period of time, the fluorescence intensity tends to decrease over time, possibly due to deterioration or decomposition of the nucleic acid probe or the fluorescent compound, and the detection sensitivity tends to decrease. According to the present invention, storage stability is improved even after a nucleic acid amplification composition containing such a fluorescently labeled probe is stored for a certain period of time, so that a decrease in fluorescence intensity can be suppressed. Therefore, it is preferable.

In one embodiment, when the composition for nucleic acid amplification of the present invention is used for detecting norovirus, the base sequence of the hybridization probe for detecting norovirus is notified by the Safety Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare ( Shokuankan No. 1105001) (SEQ ID NOS: 6 to 8), but not limited thereto. In the probe sequence, SEQ ID NO: 6 or 7 detects norovirus type G1, and SEQ ID NO: 8 detects norovirus type G2. Furthermore, if the targeted nucleic acid consists of subtypes, it may contain degenerate sequences. 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 another embodiment, when the novel coronavirus (SARS-CoV-2) is detected with the nucleic acid amplification composition of the present invention, the nucleotide sequence of the hybridization probe for its detection is published by the National Institute of Infectious Diseases. The sequences described in the "Pathogen Detection Manual 2019-nCoV" (SEQ ID NOS: 11 and 14) and the "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers published by the American Center for Disease Control and Prevention and Probes" (SEQ ID NOS: 17, 20 and 23), which can also be preferably used in the present invention, but is not limited thereto. The probe sequence detects the N region of SARS-CoV-2. Furthermore, if the targeted nucleic acid consists of subtypes, it may contain degenerate sequences. In the detection of coronaviruses such as SARS-CoV-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. do not have. 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 another embodiment, when detecting an influenza virus with the composition for nucleic acid amplification of the present invention, the base sequence of the hybridization probe for the detection is "Research Use Only CDC Influenza SARS" published by the American Centers for Disease Control and Prevention. -CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes" (SEQ ID NOS: 30, 33), etc., and can be preferably used in the present invention, but is not limited to this. do not have. In the probe sequences described above, SEQ ID NO: 30 detects influenza A and SEQ ID NO: 33 detects influenza B, but the probe sequences are not particularly limited to these. The concentration of the fluorescently labeled probe to be used is preferably 0.01 μM or more and 1.0 μM or less with respect to the whole RT-PCR reaction solution. 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.

Hybridization probes used in the present invention are preferably fluorescently labeled probes. Such fluorescently labeled probes include, for example, TaqMan hydrolysis probes (US Pat. Nos. 5,210,015, 5,538,848, 5,487,972, US Pat. Patent No. 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801), FRET hybridization probes (International Publication No. 97/46707, International Publication No. 97/46712, International Publication No. 97/46714) and the like, but are not limited thereto.

Fluorescent compounds that can be used in hybridization probes can be any known in the art, and can be selected, for example, according to the qPCR equipment that one wishes to use. Specific examples of fluorescent compounds include rhodamine (ROX) or derivatives thereof (e.g., 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA )), or rhodamine compounds such as salts thereof; fluorescein or derivatives thereof (e.g., FAM (carboxyfluorescein), JOE (6-carboxy-4′,5′-dichloro2′,7′-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5′-hexachloro-fluorescein-CE phosphoramidite)), VIC (registered trademark), BODIPY (registered trademark) series, rhodamine or derivatives thereof (for example, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA)), Cy (registered trademark) dyes (e.g., Cy3, Cy5), derivatives thereof, and non-rhodamine compounds such as salts thereof. but not limited to these. A quenching substance suitable for the fluorescent substance to be used can be used as the fluorescent compound, if necessary. Examples of quenching substances corresponding to the above fluorescent substances include TAMRA (tetramethyl-rhodamine), DABCYL (4-(4-dimethylaminophenylazo)benzoic acid), BHQ1 (BHQ: Black Hole Quencher (registered trademark) )), BHQ2, BHQ3, etc., but are not limited thereto.

The composition for nucleic acid amplification of the present invention detects the presence or absence of a target nucleic acid using a double-stranded DNA-binding fluorescent compound in place of or together with the hybridization probe as described above. good too. 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. Even when such a fluorescent compound is used, the presence or absence of the target nucleic acid can be detected by monitoring the fluorescence signal, and the labor for analysis can be reduced.

The composition for nucleic acid amplification of the present invention may contain a metal ion having a valence of 2 or more. The divalent or higher metal ion can be any metal ion known in the art, such as divalent, trivalent, tetravalent metal ions. Preferably, the divalent or higher metal ion used in the present invention is a divalent or higher cation, more preferably a divalent or trivalent cation, and particularly a divalent cation. preferable. Containing divalent cations in this way generally has the advantage that in nucleic acid amplification reactions, high resistance to contamination can be obtained more stably, and highly sensitive detection is likely to be possible.

Divalent cations that can be used in the present invention are not particularly limited as long as the effects of the present invention are achieved, but examples include magnesium ions, manganese ions, calcium ions, copper ions, iron ions, nickel ions, and zinc ions. can be done. Preferably, magnesium ions and manganese ions are included as divalent cations. In the present invention, when magnesium ions, manganese ions, or the like are added to the composition for nucleic acid amplification, magnesium or manganese may be added, or salts thereof may be added. Examples of magnesium or salts thereof include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate, etc. Examples of manganese or salts thereof include manganese, manganese chloride, manganese sulfate, manganese acetate, and the like. Magnesium, manganese, or a salt thereof is preferably added to the composition for nucleic acid amplification in an amount of about 1 to 10 mM.

In a specific embodiment, when the nucleic acid amplification composition of the present invention is a nucleic acid amplification composition for a single-enzyme RT-PCR reaction, manganese or It preferably contains a salt thereof. In a specific embodiment, the concentration of manganese or a salt thereof is preferably adjusted to contain 1 mM or more of manganese or a salt thereof in the RT-PCR reaction solution, and the concentration is adjusted to contain 1.5 mM or more of manganese or a salt thereof. More preferably, the concentration is adjusted to contain 2.0 mM or more of manganese or a salt thereof. In the case of a nucleic acid amplification composition for a two-enzyme RT-PCR reaction or for a PCR reaction without reverse transcription reaction, magnesium or a salt thereof is included from the viewpoint that high sensitivity can be obtained stably. is preferred. In a specific embodiment, the concentration of the PCR reaction solution is preferably adjusted to contain 1 mM or more of magnesium or a salt thereof, and the concentration is adjusted to contain 1.5 mM or more of magnesium or a salt thereof. More preferably, the concentration is adjusted to contain 2.0 mM or more of magnesium or a salt thereof.

The composition for nucleic acid amplification of the present invention may contain a buffer. As a buffer that can be used in the present invention, any buffer known in the art can be used, without particular limitation, Tris, Tricine, Bis-Tricine, Bicine (Bicine) and the like. The buffer may be adjusted to pH 6-9, more preferably pH 7-9, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like. The concentration of the buffering agent in the composition for nucleic acid amplification of the present invention is not particularly limited as long as the effect of the present invention is exhibited, but it can be, for example, approximately 10 to 200 mM, more preferably approximately 20 to 150 mM. At this time, it is preferable to add a salt solution in order to obtain suitable ionic conditions for the reaction. Salt solutions include, but are not limited to, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, and the like.

The composition for nucleic acid amplification of the present invention may contain an anti-DNA polymerase antibody. By including an anti-DNA polymerase antibody, for example, in a nucleic acid amplification reaction such as a PCR reaction, the enzymatic activity of the DNA polymerase can be suppressed until it is exposed to a high temperature. A PCR method can be performed. Any anti-DNA polymerase antibody known in the art can be used, and can be appropriately selected and used according to the type of DNA polymerase contained in the composition for nucleic acid amplification reaction. The concentration of the anti-DNA polymerase antibody in the composition for nucleic acid amplification of the present invention is not particularly limited, but is, for example, about 0.001 to 0.1 μg/μL, preferably about 0.005 to 0.05 μg/μL. obtain.

The composition for nucleic acid amplification of the present invention may further contain additives in addition to the above components. For example, when the composition for nucleic acid amplification of the present invention is a reagent for PCR or RT-PCR reaction, preferred examples of additives include quaternary selected from the group consisting of ammonium salts (hereinafter referred to as "betaine-like quaternary ammonium"), polypeptides (bovine serum albumin, sericin, blocking peptide fragment (hereinafter also referred to as BPF), gelatin), glycerol, glycols and surfactants; can be at least one, and so on.

Polypeptides that can be used in the present invention are preferably, for example, polypeptides with a molecular weight of about 5-500 kDa, more preferably polymerases with a molecular weight of about 6-400 kDa. In this specification, when molecular weights are indicated, they refer to values determined using SDS-PAGE, unless clearly indicated otherwise. The measurement of molecular weight by SDS-PAGE can be carried out using commercially available molecular weight markers and the like using methods and devices commonly used in the field. For example, a "molecular weight of 50 kDa" refers to a range in which a person skilled in the art normally determines that there is a band at the position of 50 kDa when the molecular weight is measured by SDS-PAGE. Also, the polypeptide used in the present invention may be a mixture of polypeptides within the above molecular weight range.

The polypeptide that can be used in the present invention is not particularly limited as long as the effect of the present invention is exhibited, and refers to a protein formed by connecting a plurality of amino acids via peptide bonds. In addition, the polypeptide used in the present invention may be, for example, a heat-denatured polypeptide (eg, gelatin) whose three-dimensional structure is unfolded by heat denaturation or the like, as long as it has a polypeptide structure in which amino acids are linked. may Specifically, polypeptides that can be used in the present invention include, for example, albumin (eg, bovine serum albumin, lactalbumin, human serum albumin, egg-derived albumin), gelatin (eg, fish gelatin, porcine gelatin), sericin, Naturally derived proteins (naturally derived polypeptides) such as casein and fibroin; blocking peptide fragments (hereinafter also referred to as BPF), collagen hydrolysates, polypeptones, yeast extracts, beef extracts, etc. artificially produced by synthesis/decomposition Polypeptides and the like can be used. More preferably, the polypeptide that can be used in the present invention is bovine serum albumin, gelatin, blocking peptide fragment (hereinafter BPF), and/or sericin. Bovine serum albumin and gelatin (especially fish gelatin) are more preferably used from the viewpoint that even a small amount is beneficial in the nucleic acid amplification reaction. These polypeptides may be used alone or in combination of two or more. Moreover, these polypeptides may be prepared by means of extraction from nature, synthesis, or the like, and commercially available products can also be suitably used.

In the present invention, when the polypeptide as described above is used, the amount thereof is not particularly limited. An amount such that the final concentration in the RT-PCR reaction solution is 0.0001 to 200 mg/mL, preferably 0.01 to 150 mg/mL, more preferably 0.1 to 130 mg/mL, still more preferably 0.5 The amount may be adjusted to ˜100 mg/mL. In addition, the amount of such polypeptides to be used may vary depending on the type of polypeptide to be used, the type and degree of desired effect, and the like. In the case of , the following usage amounts can be exemplified.
- When bovine serum albumin is used: The final concentration in the PCR reaction solution is, for example, 0.5 mg/mL or higher, preferably 1 mg/mL or higher, more preferably 2 mg/mL or higher, and still more preferably 3 mg/mL or higher. Although the upper limit is not particularly limited, it can be, for example, 10 mg/mL or less.
- When using gelatin: final concentration in the PCR reaction solution is, for example, 0.1 mg/mL or higher, preferably 1 mg/mL or higher, more preferably 5 mg/mL or higher, even more preferably 7.5 mg/mL or higher, and even more preferably is greater than or equal to 15 mg/mL. Although the upper limit is not particularly limited, it can be, for example, 50 mg/mL or less.
- When sericin is used: The final concentration in the PCR reaction solution is, for example, 1 mg/mL or higher, preferably 5 mg/mL or higher, more preferably 10 mg/mL or higher, even more preferably 20 mg/mL or higher, and even more preferably 50 mg/mL. that's all. Although the upper limit is not particularly limited, it can be, for example, 100 mg/mL or less.
- When using BPF: final concentration in the PCR reaction solution is, for example, 1 mg/mL or more, preferably 5 mg/mL or more, more preferably 10 mg/mL or more, still more preferably 20 mg/mL or more, and even more preferably 30 mg/mL that's all. Although the upper limit is not particularly limited, it can be, for example, 50 mg/mL or less.

Surfactants that can be used in the present invention include Triton X-100, Triton X-114, Tween 20, Nonidet P40, Brij35, Brij58, SDS, CHAPS, CHAPSO. , Emulgen 420 and the like, but are not particularly limited. The concentration of the surfactant in the composition for nucleic acid amplification is also not particularly limited, but may be preferably 0.0001% or higher, more preferably 0.002% or higher, and still more preferably 0.005% or higher. Although the upper limit is not particularly limited, it can be 0.1% or less as an example.

Betaine-like quaternary ammonium that can be used in the present invention includes, but is not limited to, betaine (trimethylglycine), carnitine, and the like. The betaine structure is a stable compound with both positive and negative charges in the molecule, and exhibits surfactant-like properties, and is thought to cause destabilization of the virus structure. In addition, it is known to facilitate nucleic acid amplification of DNA polymerases. Preferred said betaine-like quaternary ammonium concentration is between 0.1M and 2M, more preferably between 0.2M and 1.2M.

Furthermore, it can be combined with substances known in the art to promote PCR reactions or RT-PCR reactions. 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, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), Examples include, but are not limited to, polyethylene glycol. In order to further reduce reaction inhibition, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane- A chelating agent such as N,N,N',N'-tetraacetic acid (BAPTA) may be included.

The composition for nucleic acid amplification of the present invention can be provided in any manner. As a preferred example, the composition for nucleic acid amplification of the present invention can be provided as a premix type reagent for nucleic acid amplification. A premix type reagent for nucleic acid amplification is a sample in which substantially all of the components necessary for a nucleic acid amplification reaction are premixed, and is usually a template sample (for example, a test sample or a synthetic nucleic acid such as an internal control). A reagent for nucleic acid amplification that is ready to carry out a nucleic acid amplification reaction by simply mixing with. Such a premix type is also called a ready to use reagent. A premixed type nucleic acid amplification reagent is preferable because the composition for nucleic acid amplification is not provided separately for each part as in the prior art, and the trouble of preparing the reagent before use is unnecessary. According to the present invention, the storage stability of the composition for nucleic acid amplification is improved, deterioration in nucleic acid amplification performance is suppressed even when stored for a certain period of time at a predetermined temperature, and deterioration of the composition for nucleic acid amplification is exhibited. Since coloring is also suppressed, it is suitable as such a premix type reagent for nucleic acid amplification.

The composition for nucleic acid amplification of the present invention as described above can be used in any nucleic acid amplification reaction capable of amplifying one or more target nucleic acids. A "target nucleic acid" in the present invention may be a nucleic acid region intended to be detected by nucleic acid amplification. For example, when testing for the presence of nucleic acid derived from a pathogenic microorganism, it may be a region intended for amplification in the genomic nucleic acid of that pathogenic microorganism. Moreover, when the PCR reaction solution contains an internal control or the like, the target nucleic acid may be a region intended to be amplified in the internal control nucleic acid. 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, four, or five or more. Although the upper limit of the number of targets is not particularly limited, it can be set to 10 or less, for example. Such a PCR reaction using two or more regions as target nucleic acids is called multiplex PCR (or multiplex RT-PCR), and can be preferably carried out in the present invention.

In one preferred embodiment, the method for examining the presence or absence of a target nucleic acid using the nucleic acid amplification composition of the present invention can be a PCR reaction including at least the following steps.
(1) a step of mixing a sample containing a nucleic acid with the composition for nucleic acid amplification of the present invention;
(2) a step of conducting a PCR reaction after sealing the reaction vessel; and (3) a step of detecting the target nucleic acid with a fluorescent compound.
Here, in the step (1), the PCR reaction solution may be an RT-PCR reaction solution in which a reverse transcription reaction is also performed before the PCR reaction. Further, the step (2) may be an RT-PCR reaction accompanied by a reverse transcription reaction before the PCR reaction. When the RT-PCR reaction is performed in the step (2), even if it is a two-enzyme reaction system containing both a reverse transcriptase and a DNA polymerase, it is a one-enzyme reaction system containing a thermostable DNA polymerase having reverse transcription activity. may The steps (1) to (3) are preferably carried out in the same container. That is, it is preferable not to transfer all or part of the mixture to another container during any of the steps (1) to (3). Furthermore, in step (2), preferably in both steps (2) and (3), it is preferable not to open and close the lid of the reaction vessel after sealing the reaction vessel. In addition, the sample containing nucleic acid used in the step (1) may be a sample that may contain contaminants and insoluble substances, such as a suspension previously suspended in water or a buffer solution, or a stool sample. A solid sample such as may be added directly to the PCR reaction solution.

The subject to be tested in the testing method using the composition for nucleic acid amplification of the present invention may be, for example, a nucleic acid derived from a pathogenic microorganism, but is not particularly limited. Here, pathogenic microorganisms refer to any microorganisms that can infect living organisms such as mammals including humans and cause infectious diseases, and are not limited to prokaryotes and eukaryotes such as bacteria or fungi, This concept also includes viruses and the like.

Pathogenic microorganisms include Escherichia coli (e.g. enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), enterohemorrhagic Escherichia coli (EHEC)). Campylobacter, Welsh, Salmonella, Listeria, Botulinum, Cereus, Pseudomonas (multidrug-resistant Pseudomonas aeruginosa), Clostridium, Legionella, Streptococcus, yellow Staphylococci (e.g., methicillin-resistant Staphylococcus aureus), Acinetobacter (e.g., drug-resistant Acinetobacter), Vibrio parahaemolyticus, Shigella, Diphtheria, Vibrio cholerae, Pertussis, Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Examples include Rickettsia, Chlamydia, Coxiella, Toxoplasma, but are not particularly limited. In particular, the detection of pathogenic bacteria that cause food poisoning, such as salmonella, enterohemorrhagic Escherichia coli (EHEC), and shigella, is often performed as an intestinal bacteria test that handles a large number of samples, and is highly workable. The simple inspection method of the present invention is particularly useful.

In certain embodiments, pathogenic microorganisms may be viruses. Viruses targeted by the present invention may be DNA viruses or RNA viruses. DNA viruses include Herpesviridae viruses (e.g., herpes simplex virus, cytomegalovirus); Adenoviridae viruses (e.g., human adenovirus); Hepadnaviridae viruses (e.g., hepatitis B virus); Papovaviridae viruses (e.g., papillomavirus, polyomavirus); Parvoviridae virus (e.g., adeno-associated virus, human parvovirus); Asphaviridae virus (e.g., African swine fever virus), etc., but are particularly limited. not a thing

The RNA virus may be either a non-enveloped RNA virus derived from a lipid bilayer membrane or an enveloped RNA virus. Such non-enveloped RNA viruses include Astroviridae viruses (e.g., Astroviruses); Caliciviridae viruses (e.g., Sapoviruses, Noroviruses); Picornaviridae viruses (e.g., Hepatitis A virus, Echovirus, enterovirus, coxsackievirus, poliovirus, rhinovirus); hepeviridae viruses (e.g., hepatitis E virus); reoviridae viruses (e.g., rotavirus), etc., but are preferably It is useful for detecting Caliciviridae viruses and Reoviridae viruses, more preferably for detecting norovirus, sapovirus, and rotavirus, and particularly useful for detecting norovirus.

Noroviruses have a capsid structure in which the viral RNA genome is enclosed inside an icosahedron consisting of capsid proteins of approximately 30 nm. The capsid structure is resistant to inactivation by gastric acid and surfactant action of bile acid so that the virus can survive in harsh environments such as the gastrointestinal tract. Ordinary surfactants and virus-inactivating agents such as 70% ethanol cannot destroy this capsid structure, and virus infectivity is maintained. In order to destroy the capsid structure, heat treatment under severe conditions of at least 85°C or more and 1 minute or more is required, but it is very difficult to extract and purify RNA using a commercially available viral RNA extraction kit. labor intensive.

The main cause of norovirus infection is eating and drinking food contaminated with norovirus, but since infection is often transmitted through human hands, regular stool tests are required at cooking facilities, medical sites, nursing homes for the elderly, nursery schools, etc. is required. The Hygiene Management Manual for Mass Cooking Facilities adds that the stool examination of cooking workers should include norovirus testing at least once a month or, if necessary, from October to March, which is the epidemic season for norovirus. . This is because there are quite a few people who are infected with the virus but have no symptoms (healthy carriers), and these people may unknowingly spread the infection. In addition, cooks with symptoms such as diarrhea and vomiting should consult a medical institution, and if they are found to be infected with norovirus, highly sensitive tests such as real-time PCR will be performed to confirm that they do not have norovirus. Appropriate measures, such as refraining from cooking work that comes in direct contact with food, should be taken until this is confirmed. Therefore, it is of great significance to conduct regular examinations, including those of healthy individuals, and it is required to conduct examinations of many specimens. It is particularly preferable that the present invention be used for testing for norovirus from the viewpoint that testing can be performed easily and with good workability even when handling a large number of samples in this way, and the burden on the person in charge of testing can be reduced.

Examples of 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, Chikungunya virus); Coronaviridae viruses (e.g., , SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus); Orthomyxoviridae viruses (e.g., influenza virus); Rhabdoviridae viruses (e.g., rabies virus); fever virus, hantavirus); Paramyxoviridae viruses (eg, measles virus, human respiratory syncytial virus); Filoviridae viruses (eg, Ebola virus), etc., but are not particularly limited. Preferably, it is useful for detecting coronaviruses and orthomyxoviridae viruses, especially coronaviruses (SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus) and influenza viruses (influenza virus type A, B type, type C).

Coronaviruses are causative viruses that cause respiratory infections including colds, and about 10 to 35% of 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) -CoV-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.

In the mutated coronavirus SARS-CoV-2, which was confirmed to occur in Wuhan City, Hubei Province, China in 2019, a test method using nucleic acid amplification technology was established as soon as the analysis of viral genome RNA was completed (for example, , Non-Patent Document 1, Non-Patent Document 2). Also in Japan, a method for detecting SARS-CoV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases (Non-Patent Document 4). Currently, rapid one-step RT-PCR without RNA extraction and purification steps is available from biological and environmental swab samples such as pharyngeal/nasal swabs, saliva, sputum, and fecal samples containing coronaviruses, especially SARS-CoV-2. methods have been developed to detect In addition, from the viewpoint of preventing infection and preventing the spread of infection, the expansion of testing, including asymptomatic patients, has greatly increased the frequency of testing, raising concerns about an increased burden on test personnel. Under such circumstances, since the PCR reaction solution needs to be prepared on demand, the workload of preparing the reaction solution in high-frequency testing is heavy, and the development of a testing method with improved workability is strongly desired. The present invention is an excellent method that improves workability and operability by improving the storage stability of the PCR reaction solution, and can detect the target nucleic acid with high sensitivity. It is particularly useful in testing for SARS-CoV-2 virus.

Influenza viruses, like coronaviruses, are causative viruses that cause respiratory infections. In addition to frequently causing serious respiratory diseases, it is known to cause complications such as bronchitis, pneumonia, otitis media, and acute encephalopathy. There are three main types of influenza, A, B, and C, with types A and B spreading epidemics every year. Two types of glycoproteins called hemagglutinin (HA) and neuraminidase (NA) are present on the surface of influenza virus particles and are involved in human infection. There are 15 different subtypes of HA and 9 different subtypes of NA, and viruses with various combinations of these are widely distributed. Due to the regular emergence of combinations of different subtypes of the two glycoproteins described above, influenza A epidemics occur every few years to decades due to mutated viruses worldwide.

Influenza virus and SARS-CoV-2 coronavirus are known to cause similarly severe respiratory illness and have very similar symptoms. Because treatment methods differ for each virus, it is necessary to identify the causative virus at an early stage. INDUSTRIAL APPLICABILITY The present invention can detect a plurality of target nucleic acids with good workability and operability with sufficient sensitivity even when target nucleic acids at two or more sites derived from two or more pathogenic microorganisms are detected simultaneously. Therefore, the present invention simultaneously tests influenza viruses and coronaviruses (coronaviruses that are likely to cause serious illness such as SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus), especially SARS-CoV-2 virus and influenza virus. useful in some cases.

Samples used in the present invention include, for example, pharyngeal swabs, nasal swabs, sputum, feces (fecal excretion, rectal stool), vomit, saliva, gargle, tears, and the like, but are particularly limited. Instead, it can be used for all substances derived from living organisms. In particular, it is useful for detection from feces, pharyngeal swab, nasal swab, sputum, lung aspirate, cerebrospinal fluid, gargle, saliva, tears, cultured cells, and culture supernatant. It is useful for detecting saliva samples, sputum samples, gargles, tears, throat swabs, nasal swabs. In the present invention, RNA may be isolated from these samples using a commercially available RNA purification kit or the like, and may be provided as a sample. Therefore, it may be a sample obtained by suspending the sample in water, physiological saline, or a buffer solution. Alternatively, the sample may be directly subjected to detection. 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.

In the step (1), when using a sample that may contain contaminants and insoluble substances, a suspension previously suspended in water or a buffer solution, or a solid sample such as a fecal sample, pathogenic microorganisms In order to facilitate the extraction of RNA or DNA from rigid structures such as capsids and cell walls, the sample may be a sample mixed with a solution containing a buffer solution, an acid or alkaline solution, an organic solvent, or the like. The acidic solution contained in the solution is not particularly limited as long as it is an acidic solution. Examples of acidic solutions include aqueous formic acid, aqueous acetic acid, aqueous butyric acid, aqueous hydrochloric acid, aqueous nitric acid, aqueous sulfuric acid, aqueous citric acid, aqueous lactic acid, aqueous phosphoric acid, aqueous benzoic acid, aqueous oxalic acid, aqueous tartaric acid, and aqueous ascorbic acid. , an aqueous sulfonic acid solution, etc., and can be used singly or in combination of two or more. The alkaline solution is not particularly limited as long as it is an alkaline solution. Examples of alkaline solutions include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate. Aqueous solutions, Tris buffers, glycine buffers, phosphate buffers, borate buffers, Good's buffers (TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPS) and the like, one type alone Or it can be used in combination of two or more. Examples of organic solvents include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphorictriamide, dimethylsulfoxide, acetone, acetonitrile, ethanol, and methanol. , 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Furthermore, the sample added in the step (1) may be subjected to heat treatment after being mixed with the solution. The conditions for the heat treatment are not particularly limited, but may be 60° C. or higher, preferably 70° C. or higher, more preferably 80° C. or higher, and still more preferably 90° C. or higher for 1 second or longer.

A sample of another aspect of the present invention is a swab test sample. Wiping inspections are useful for clarifying the contamination route and grasping the contamination status of the facility environment. In the present invention, the swab 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. . As a specific procedure for the swab test, "Improvement of the norovirus test method for swab samples" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html) is exemplified. 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. Since these test samples contain a large amount of dirt and dust at the test site, this technique with enhanced contamination resistance in samples that can contain contaminants and insoluble substances is beneficial for these tests.

In certain embodiments, the methods of the invention may use samples that have not undergone nucleic acid isolation procedures. For example, the present invention can be used to isolate nucleic acids from various samples using commercially available nucleic acid purification kits, or to perform prior heat treatment to expose genomic nucleic acids from structures of pathogenic microorganisms (e.g., cell membranes, capsid structures), etc. Untreated, untreated samples can be used. It is preferable to use these untreated samples from the viewpoint of convenience because no troublesome pretreatment is required. Alternatively, it may be a sample that has undergone nucleic acid extraction without separation and purification to remove contaminants. Here, the sample subjected to nucleic acid extraction without separation and purification means that the nucleic acid is exposed in the sample. Extraction and exposure of encapsulated nucleic acids (however, cell membranes, capsids, envelope fragments, etc. remaining after disruption should not be removed). According to the present invention, nucleic acids can be amplified satisfactorily even in samples prepared without such isolation and purification, and test results can be stably obtained. Such a nucleic acid extraction treatment without separation and purification can be performed prior to the step (1).

Contaminants and insoluble substances that may be contained in the sample subjected to PCR or RT-PCR in the step (1) include feces (feces, rectal feces), vomit, saliva, sputum, gargle, nasal swab, pharynx Examples include those derived from wipes, tears, blood, and swab test samples, but are not limited to those derived from living organisms, and can be used for environmental test samples in general, especially feces (excretion stool, rectal stool), saliva, sputum, gargle, tear, pharyngeal swab, and nasal swab. The concentration of insoluble substances that can be contained varies depending on the test sample, but if it is contained in the PCR reaction solution or RT-PCR reaction solution at a turbidity of OD660, for example, 0.01 Abs / μL or more, the test sensitivity may be affected. There is, but is not limited to. For example, the turbidity OD660 may be 0.05 Abs/μL or more, 0.1 Abs/μL or more, 0.5 Abs/μL or more, or 1 Abs/μL or more, but is not particularly limited. As long as the effect of the present invention is exhibited, the upper limit of the concentration of the insoluble substance that can be contained is not particularly limited. According to the present invention, it may be possible to detect the target nucleic acid even if such a highly turbid test sample is subjected to a PCR reaction containing an antioxidant.

The PCR cycle in the step (2) includes: 1. 2. heat treatment; A reverse transcription reaction step may be included. Moreover, before and after each step, a heat treatment step for activating the hot start enzyme may be included. One heat treatment step can include disrupting the virus to expose the nucleic acid within the virus and/or activating hot start enzymes in a nucleic acid amplification reaction. The temperature and time of the heat treatment step may be 60° C. or higher and 1 second or longer, preferably 70° C. for 30 seconds or longer, more preferably 80° C. for 30 seconds or longer, and particularly preferably 85° C. for 30 seconds or longer. seconds or more. The temperature of the reverse transcription reaction in 2 is determined by the reverse transcription activity of the reverse transcriptase used 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 3, [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 15 seconds or less, more preferably 10 seconds or less. It is desirable to set up a measurement program for As used herein, the term "PCR extension time" refers to the set temperature of the thermal cycler.

As shown by the results of test examples described later, the coexistence of an antioxidant can improve the storage stability of nucleic acid amplification reagents containing DNA polymerase and the like. Therefore, from another aspect, the present invention provides (1) a DNA polymerase, (2) at least one nucleotide selected from the group consisting of oligonucleotides and mononucleotides, (3) a divalent or higher metal ion, and ( 4) A method for improving the storage stability of a nucleic acid amplification composition by allowing (5) an antioxidant to coexist with a nucleic acid amplification composition containing a buffer is also provided.
The types and amounts of antioxidants used in this embodiment, the types and amounts of DNA polymerase, the types and amounts of nucleotides such as primers and probes, the types of samples used for testing, pathogenic microorganisms to be tested, etc. , may be the same as those described in detail in the composition for nucleic acid amplification.

In the present invention, the method for determining the storage stability of the composition for nucleic acid amplification is not limited, but it can be determined from the amount of nucleic acid amplification after storage under predetermined conditions for a predetermined period of time. For example, it can be confirmed by subjecting the amplified product after RT-PCR to electrophoresis and comparing it with a band estimated from the base number of the template RNA. In electrophoresis, it is possible to qualitatively determine the amount of amplified nucleic acid 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, it is possible to determine the amount of amplified nucleic acid from the height of the peak in the melting curve. Further, 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. Therefore, the amount of amplified nucleic acid can be determined by comparing the reaching fluorescence intensity and increasing/decreasing the Ct value.

Another method for determining the storage stability of a composition for nucleic acid amplification is the change in color tone of the composition over time. Constituent substances such as some metal ions, oligonucleotides, amino acids, peptides, proteins, sugars and sugar alcohols contained in the PCR reaction solution may be subject to deterioration or decomposition of the constituent substances themselves or the Maillard reaction that occurs between the constituent substances. It is known that the color tone changes and becomes colored over time due to the chemical reaction of . A colored solution has an absorption spectrum in at least one wavelength band. The color of the solution is classified by the maximum absorption wavelength, and the color tones observed in the present invention (maximum absorption wavelength in parentheses) are greenish yellow (400-435 nm), yellow (435-480 nm), orange (480-490 nm), red (490-500 nm), red-violet (500-560 nm), purple (560-580 nm), blue (580-595 nm), green-blue (595-610 nm), blue-green (610-750 nm), green (750-800 nm) , white to black shades are also included, but are not particularly limited as long as they are visually perceptible. Determination of color tone change is not particularly limited, but can be visually identified with the naked eye. In a preferred embodiment, the determination can be made based on changes in measured values using various measuring devices such as a color difference meter, colorimeter, spectrophotometer, and illuminance meter. When using measured values from various measuring devices, determination can be made by absolute evaluation and relative comparison of measured values before and after storage. For example, when using spectrophotometer measurements, a change in color tone can be evaluated as a change in absorbance. The wavelength of absorbance to be measured is not particularly limited because it varies depending on the color tone of the solution, but at least one or more wavelengths are selected.

In one preferred embodiment, color change before and after storage may be evaluated according to the method described in Test Example 4 below. Specifically, it is preferable to measure the absorbance in the region of 435 to 490 nm before and after storage using a spectrophotometer (optical path length 10 nm) to evaluate the change in color tone over time. For example, the absorbance is measured at intervals of 5 nm (e.g., 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490 nm) in the region of 435 to 490 nm, and the composition for nucleic acid amplification is measured at each absorbance. By comparing the absorbance values of the object before and after storage, the change in color tone over time can be evaluated.

Storage conditions (also referred to as storage conditions) for the nucleic acid amplification composition of the present invention are not particularly limited. The storage temperature is, for example, −90° C. or higher and 50° C. or lower, more preferably −30° C. or higher and 40° C. or lower, still more preferably 0° C. or higher and 30° C. or lower, still more preferably 4° C. or higher and 25° C. or lower. The storage period is 1 minute or longer, preferably 1 hour or longer, more preferably 12 hours or longer, still more preferably 1 day or longer, and even more preferably 4 days or longer. In the present invention, even after storage for 7 days or 14 days, deterioration in the performance of the nucleic acid amplification reaction could be suppressed, and even after storage at room temperature for 4 days, change in color tone was suppressed and it was stable, which will be described later. It is confirmed in the results of the test example. Although the upper limit is not particularly limited, it can be, for example, within one year.

In a specific preferred embodiment, the nucleic acid amplification composition of the present invention exhibits suppressed deterioration in nucleic acid amplification reaction performance even when stored at 4° C. for 4 to 14 days, and after storage under these conditions, the nucleic acid amplification reaction is not affected. The composition for nucleic acid amplification is capable of amplifying and detecting a target nucleic acid with sufficient sensitivity when used.

In yet another preferred embodiment, the nucleic acid amplification composition of the present invention exhibits suppressed deterioration in nucleic acid amplification reaction performance even when stored at 25° C. for 4 to 7 days, and the nucleic acid amplification reaction performance is suppressed after storage under the conditions. The composition for nucleic acid amplification is capable of amplifying and detecting a target nucleic acid with sufficient sensitivity even when used in Since storage at 25° C. also corresponds to an accelerated test of refrigerated storage conditions, the composition of the present invention may be a stable composition even when refrigerated for a longer period of time.

In another preferred embodiment, the composition for nucleic acid amplification of the present invention is a nucleic acid in which a change in color tone measured by any absorbance in the 435 to 490 nm region is suppressed when left at 25° C. for 4 days. A composition for amplification. The composition of the present invention can suppress color change at any absorbance within the range of 435-490 nm. When nucleic acid amplification is detected using the luminescence or quenching phenomenon of a fluorescent compound, a change in color tone of the composition for nucleic acid amplification may affect the measurement. Therefore, the composition for nucleic acid amplification of the present invention is also beneficial in that it can be a reagent capable of stably detecting nucleic acid amplification even after being stored for a certain period of time.

Another aspect of the present invention is a test kit or composition for detecting nucleic acids derived from viruses or pathogenic microorganisms in a sample, characterized by comprising a PCR reaction reagent containing an antioxidant. , a test kit or composition for performing a PCR method with a fluorescent compound.

The types and amounts of antioxidants used in these embodiments, the types and amounts of DNA polymerases, the types and amounts of nucleotides such as primers and probes, the types of samples used for testing, pathogenic microorganisms to be tested, etc. may be the same as those detailed in the inspection method above.

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

（２）反応液の調製および抗酸化剤の添加
反応液（１０ｘＢｕｆｆｅｒを１ｘとなるように希釈して使用）
（ｒＴａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ １０ｘＢｕｆｆｅｒ（東洋紡）添付品）
プライマー液（１０ｘプライマー液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
プローブ液（１０ｘプローブ液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
０．２ｍＭ ｓＮＴＰｓ Ｍｉｘｔｕｒｅ （東洋紡）
２ｍＭ Ｍｎ（ＯＡｃ）_２ （東洋紡）
４．２ｎｇ／μＬ ｒＴｔｈ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡）
０．０１μｇ／μＬ 抗Ｔｔｈ 抗体
前記各試薬を混合後、表１に記載した終濃度となるように各抗酸化剤を１．５μＬ添加し、最終液量が４９μＬとなるようにＲＴ－ＰＣＲ反応液を調製した。陰性対照は、１．５μＬのＲＮａｓｅ Ｆｒｅｅ Ｗａｔｅｒを添加した。
（３）ＰＣＲ反応液の保管
調製したＰＣＲ反応液を２５℃で４日間又は７日間、遮光下にて保管した。エリソルビン酸ナトリウムのみ４℃で７日間又は１４日間での遮光下の保管も実施した。
（４）ＰＣＲの実施
調製直後のＰＣＲ反応液および、（３）の条件にて保管していたそれぞれのＰＣＲ反応液を用いて、以下の方法にて同様にＰＣＲを実施した。各反応液に１２５０コピー／μＬに調製したノロウイルスＧ２ ＲＮＡを１μＬずつ添加し、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。ＢｉｏＲａｄ製ＣＦＸ９６ＷＥＬＬ ＤＥＥＰを使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。５２℃、４０サイクルの伸長ステップで蛍光値の読み取りを行った。次いで、ＢｉｏＲａｄ製ＣＦＸ ＭａｎａｇｅｒまたはＣＦＸ Ｍａｅｓｔｒｏソフトウエアによって算出される到達相対蛍光強度を測定値とした。
９０℃ １分（熱変性条件）
５８℃ ５分（逆転写条件）
９５℃ １秒－５２℃ １０秒 １０サイクル（ＰＣＲ）
９５℃ １秒－５２℃ １０秒 ４０サイクル（ＰＣＲ－蛍光読み取り）
（５） 結果
開始時の測定値として得られた到達相対蛍光強度を１００％とし、各条件において得られた測定値との比較を実施した。この結果を図１、２に示す。結果、陰性対照においては、２５℃７日間保管で５２％まで蛍光強度が低下してしまうが、各抗酸化剤を添加した条件においては８５％以上の蛍光強度を維持しており、保存安定性が向上していることが確認された。また、４℃１４日間の保管条件においても、陰性対照では７３％まで蛍光強度が低下してしまう反面、エリソルビン酸ナトリウムを添加した条件では９７％であり、保存安定性が向上しＰＣＲ性能が維持されていることが確認された。 Test Example 1.1 Study of addition of antioxidant to enzyme-based 1-step RT-PCR reaction solution The reaction solution having the composition shown below is used as the basic composition, and after adding the antioxidant, it is stored under each temperature condition, and then Norovirus RNA was detected in the reaction mixture in a one-enzyme, one-step RT-PCR.
(1) Preparation of Antioxidant An antioxidant was dissolved in water or dimethylsulfoxide, and the final concentration of the solution during storage in the PCR reaction solution was as shown in Table 1 below.

(2) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
Primer solution (10x primer solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
Probe solution (10x probe solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
2 mM Mn(OAc) ₂ (Toyobo)
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody After mixing each of the above reagents, 1.5 μL of each antioxidant was added so that the final concentration was as shown in Table 1, and the RT-PCR reaction was performed so that the final volume was 49 μL. A liquid was prepared. Negative controls added 1.5 μL of RNase Free Water.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 25°C for 4 days or 7 days under light shielding. Only sodium erythorbate was also stored at 4°C for 7 days or 14 days in the dark.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. 1 μL of norovirus G2 RNA adjusted to 1250 copies/μL was added to each reaction solution, and RT-PCR was performed in a 50 μL reaction system. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 40 cycles at 52°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
90°C for 1 minute (thermal denaturation conditions)
58°C for 5 minutes (reverse transcription conditions)
95°C 1 sec-52°C 10 sec 10 cycles (PCR)
95°C 1 sec - 52°C 10 sec 40 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIGS. As a result, in the negative control, the fluorescence intensity decreased to 52% after storage at 25 ° C. for 7 days, but the fluorescence intensity was maintained at 85% or more under the conditions where each antioxidant was added, indicating storage stability. was confirmed to be improving. In addition, even under storage conditions of 4 ° C. for 14 days, the fluorescence intensity decreased to 73% in the negative control, while it was 97% in the condition where sodium erythorbate was added, improving storage stability and maintaining PCR performance. It was confirmed that

（２）反応液の調製および抗酸化剤の添加
反応液（１０ｘＢｕｆｆｅｒを１ｘとなるように希釈して使用）
（ｒＴａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ １０ｘＢｕｆｆｅｒ（東洋紡）添付品）
プライマー液 （１０ｘプライマー液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
プローブ液（１０ｘプローブ液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
０．２ｍＭ ｓＮＴＰｓ Ｍｉｘｔｕｒｅ （東洋紡）
５ｍＭ ＭｇＳＯ_４
４．２ｎｇ／μＬ ｒＴｔｈ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡）
０．０１μｇ／μＬ 抗Ｔｔｈ 抗体
０．１Ｕ／μＬ ＭＭＬＶ ＲＴａｓｅ（東洋紡）
前記各試薬を混合後、表２に記載した終濃度となるように各抗酸化剤を１．５μＬ添加し、最終液量が４９μＬとなるようにＲＴ－ＰＣＲ反応液を調製した。陰性対照は、１．５μＬのＲＮａｓｅ Ｆｒｅｅ Ｗａｔｅｒを添加した。
（３）ＰＣＲ反応液の保管
調製したＰＣＲ反応液を４℃で４日間、遮光下にて保管した。
（４）ＰＣＲの実施
調製直後のＰＣＲ反応液および、（３）の条件にて保管していたそれぞれのＰＣＲ反応液を用いて、以下の方法にて同様にＰＣＲを実施した。各ＰＣＲ反応液に、１２５０コピー／μＬに調製したノロウイルスＧ２ ＲＮＡを１μＬずつ添加し、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。ＢｉｏＲａｄ製ＣＦＸ９６ＷＥＬＬ ＤＥＥＰを使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。６０℃、３０サイクルの伸長ステップで蛍光値の読み取りを行った。次いで、ＢｉｏＲａｄ製ＣＦＸ ＭａｎａｇｅｒまたはＣＦＸ Ｍａｅｓｔｒｏソフトウエアによって算出される到達相対蛍光強度を測定値とした。
４２℃ ５分（逆転写条件）
９５℃ １０秒
９５℃ ５秒－６０℃ ３０秒 １０サイクル（ＰＣＲ－蛍光読み取り）
９５℃ ５秒－６０℃ ３０秒 ３０サイクル（ＰＣＲ－蛍光読み取り）
（５） 結果
開始時の測定値として得られた到達相対蛍光強度を１００％とし、各条件において得られた測定値との比較を実施した。この結果を図３に示す。結果、陰性対照においては、４℃４日間保管で６６％まで蛍光強度が低下してしまうが、各抗酸化剤を添加した条件においては８０％以上の蛍光強度を維持しており、保存安定性が向上しＰＣＲ性能が維持されていることが確認された。 Test Example 2.2 Examination of addition of antioxidant to enzyme-based 1-step RT-PCR reaction solution The reaction solution having the composition shown below is used as a basic composition, and an antioxidant is added to each reaction solution under each condition. After storage, norovirus RNA in the reaction solution was detected in a two-enzyme one-step RT-PCR.
(1) Preparation of Antioxidant An antioxidant was dissolved in water or dimethylsulfoxide, and the final concentration of the solution during storage in the PCR reaction solution was as shown in Table 2 below.

(2) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
Primer solution (10x primer solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
Probe solution (10x probe solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
₅ mM MgSO4
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody 0.1 U/μL MMLV RTase (Toyobo)
After mixing the above reagents, 1.5 μL of each antioxidant was added so as to have the final concentration shown in Table 2, and an RT-PCR reaction solution was prepared so that the final volume was 49 μL. Negative controls added 1.5 μL of RNase Free Water.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 4°C for 4 days under light shielding.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. 1 μL of norovirus G2 RNA adjusted to 1250 copies/μL was added to each PCR reaction solution, and RT-PCR was performed in a 50 μL reaction system. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 30 cycles at 60°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 sec 95°C 5 sec - 60°C 30 sec 10 cycles (PCR-fluorescence reading)
95°C 5s-60°C 30s 30 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIG. As a result, in the negative control, the fluorescence intensity decreased to 66% after storage for 4 days at 4 ° C., but the fluorescence intensity was maintained at 80% or more under the conditions where each antioxidant was added, indicating storage stability. was confirmed to be improved and the PCR performance was maintained.

（２）反応液の調製および抗酸化剤の添加
反応液（１０ｘＢｕｆｆｅｒを１ｘとなるように希釈して使用）
（ｒＴａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ １０ｘＢｕｆｆｅｒ（東洋紡）添付品）
プライマー液 （１０ｘプライマー液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
プローブ液（１０ｘプローブ液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
０．２ｍＭ ｓＮＴＰｓ Ｍｉｘｔｕｒｅ （東洋紡）
２ｍＭ Ｍｎ（ＯＡｃ）_２ （東洋紡）
４．２ｎｇ／μＬ ｒＴｔｈ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡）
０．０１μｇ／μＬ 抗Ｔｔｈ 抗体
前記各試薬を混合後、表１に記載した終濃度となるように各抗酸化剤を１．５μＬ添加し、最終液量が４９μＬとなるようにＲＴ－ＰＣＲ反応液を調製した。陰性対照は、１．５μＬのＲＮａｓｅ Ｆｒｅｅ Ｗａｔｅｒを添加した。
（３）ＰＣＲ反応液の保管
調製したＰＣＲ反応液を４℃で４日間、遮光下にて保管した。
（４）ＰＣＲの実施
調製直後のＰＣＲ反応液および、（３）の条件にて保管していたそれぞれのＰＣＲ反応液を用いて、以下の方法にて同様にＰＣＲを実施した。各反応液に１２５０コピー／μＬに調製したノロウイルス検体を１μＬずつ添加し、５０μＬ反応系としてＲＴ－ＰＣＲを実施した。ＢｉｏＲａｄ製ＣＦＸ９６ＷＥＬＬ ＤＥＥＰを使用して、以下の温度サイクルでリアルタイムＰＣＲ反応を実施した。５２℃、４０サイクルの伸長ステップで蛍光値の読み取りを行った。次いで、ＢｉｏＲａｄ製ＣＦＸ ＭａｎａｇｅｒまたはＣＦＸ Ｍａｅｓｔｒｏソフトウエアによって算出される到達相対蛍光強度を測定値とした。
９０℃ １分（熱変性条件）
５８℃ ５分（逆転写条件）
９５℃ １秒－５２℃ １０秒 １０サイクル（ＰＣＲ）
９５℃ １秒－５２℃ １０秒 ４０サイクル（ＰＣＲ－蛍光読み取り）
（５） 結果
開始時の測定値として得られた到達相対蛍光強度を１００％とし、各条件において得られた測定値との比較を実施した。この結果を図４に示す。結果、陰性対照においては、４℃、４日間保管で８０％まで蛍光強度が低下してしまうが、各抗酸化剤を添加した条件においては９４％以上の蛍光強度を維持しており、保存安定性が向上していることが確認された。 Test Example 3.1 Examination of addition of antioxidant to enzyme system 1-step RT-PCR reaction solution 2
A reaction solution having the composition shown below was used as a basic composition, and after adding an antioxidant, it was stored under each temperature condition, and then a norovirus sample in the reaction solution was detected by 1-enzyme-system, 1-step RT-PCR.
(1) Preparation of Norovirus Specimen As a norovirus sample, a norovirus specimen, Norovirus GII Positive Control (ZeptoMetrix, intact) was used. Each sample was prepared to contain G2 norovirus, 1250 copies/μL.
(2) Preparation of Antioxidant An antioxidant was dissolved in water or dimethylsulfoxide, and the final concentration of the solution during storage in the PCR reaction solution was as shown in Table 3 below.

(2) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
Primer solution (10x primer solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
Probe solution (10x probe solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
2 mM Mn(OAc) ₂ (Toyobo)
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody After mixing each of the above reagents, 1.5 μL of each antioxidant was added so that the final concentration was as shown in Table 1, and the RT-PCR reaction was performed so that the final volume was 49 μL. A liquid was prepared. Negative controls added 1.5 μL of RNase Free Water.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 4°C for 4 days under light shielding.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. 1 μL of a norovirus sample adjusted to 1250 copies/μL was added to each reaction solution, and RT-PCR was performed using a 50 μL reaction system. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 40 cycles at 52°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
90°C for 1 minute (thermal denaturation conditions)
58°C for 5 minutes (reverse transcription conditions)
95°C 1 sec-52°C 10 sec 10 cycles (PCR)
95°C 1 sec - 52°C 10 sec 40 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIG. As a result, in the negative control, the fluorescence intensity decreased to 80% after storage at 4 ° C. for 4 days, but the fluorescence intensity of 94% or more was maintained under the condition of adding each antioxidant, and the storage stability. improved performance was confirmed.

（２）反応液の調製および抗酸化剤の添加
反応液（１０ｘＢｕｆｆｅｒを１ｘとなるように希釈して使用）
（ｒＴａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ １０ｘＢｕｆｆｅｒ（東洋紡）添付品）
プライマー液 （１０ｘプライマー液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
プローブ液（１０ｘプローブ液を１ｘとなるように希釈して使用）
（ノロウイルス検出キットＧ１／Ｇ２－高速プローブ検出－（東洋紡）添付品）
０．２ｍＭ ｓＮＴＰｓ Ｍｉｘｔｕｒｅ （東洋紡）
２ｍＭ Ｍｎ（ＯＡｃ）_２ （東洋紡）
４．２ｎｇ／μＬ ｒＴｔｈ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡）
０．０１μｇ／μＬ 抗Ｔｔｈ 抗体
前記各試薬を混合後、表１に記載した終濃度となるように各抗酸化剤を１．５μＬ添加し、最終液量が４９μＬとなるようにＲＴ－ＰＣＲ反応液を調製した。陰性対照は、１．５μＬのＲＮａｓｅ Ｆｒｅｅ Ｗａｔｅｒを添加した。
（３）ＰＣＲ反応液の保管
調製したＰＣＲ反応液を２５℃で４日間、遮光下にて保管した。
（４）吸光度の測定
分光光度計（光路長１０ｍｍ、日立ハイテクサイエンス社製）を利用し、各反応液の吸光度を測定した。反応液は保管後に黄色～茶褐色へと変化するため、対応する色調の吸光度領域４３５ｎｍ～４９０ｎｍを５ｎｍ間隔で測定した。
（５）結果
保管０日目と保管４日目のそれぞれの吸光度の測定値の差分を算出し、色調の変化値（ΔＡｂｓ）として表５に記載した。陰性対照の色調の変化値を基準（１００％）とし、各抗酸化物質を添加した条件における色調の変化値を比較した（表６）。結果、エリソルビン酸ナトリウムおよび亜硫酸ナトリウムのいずれの抗酸化剤を用いた場合でも色調の変化の値は１００％未満となっており、反応液の色調の変化が抑えられていることを確認した。

Test example 4. Addition of antioxidant to RT-PCR reaction solution and color change after storage Absorbance measurements were performed.
(1) Preparation of Antioxidant An antioxidant was dissolved in water, and the final concentration in the PCR reaction solution during storage was as shown in Table 4 below.

(2) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
Primer solution (10x primer solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
Probe solution (10x probe solution diluted to 1x)
(Norovirus detection kit G1/G2-High-speed probe detection-(Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
2 mM Mn(OAc) ₂ (Toyobo)
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody After mixing each of the above reagents, 1.5 μL of each antioxidant was added so that the final concentration was as shown in Table 1, and the RT-PCR reaction was performed so that the final volume was 49 μL. A liquid was prepared. Negative controls added 1.5 μL of RNase Free Water.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 25°C for 4 days under light shielding.
(4) Measurement of Absorbance Using a spectrophotometer (optical path length 10 mm, manufactured by Hitachi High-Tech Science), the absorbance of each reaction solution was measured. Since the reaction solution changed from yellow to dark brown after storage, the absorbance region of the corresponding color tone from 435 nm to 490 nm was measured at intervals of 5 nm.
(5) Results The difference between the measured absorbance values on the 0th day of storage and the 4th day of storage was calculated and shown in Table 5 as a color tone change value (ΔAbs). Using the color change value of the negative control as a standard (100%), the color change values under the condition of adding each antioxidant were compared (Table 6). As a result, the change in color tone was less than 100% when either sodium erythorbate or sodium sulfite was used as an antioxidant, confirming that the change in color tone of the reaction solution was suppressed.

Test Example 5.2 Enzyme system 1 step Examination of addition of antioxidant to multiplex RT-PCR reaction solution 1
The reaction solution with the composition shown below is the basic composition, and an antioxidant is added to each reaction solution, stored under each condition, and then in a two-enzyme system 1-step RT-PCR, SARS-CoV- Two target nucleic acid regions (N1 region, N2 region) in 2RNA were detected.
(1) Preparation of Antioxidant Antioxidant was dissolved in water, and the final concentration in the PCR reaction solution during storage was as shown in Table 7 below.

(2) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
Primer/probe solution (10x primer solution diluted to 1x)
(SARS-CoV-2 Detection Kit -Multi- (Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
₅ mM MgSO4
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody 0.1 U/μL MMLV RTase (Toyobo)
After mixing the above reagents, 1.5 μL of each antioxidant was added so as to have the final concentration shown in Table 7, and an RT-PCR reaction solution was prepared so that the final volume was 49 μL. Negative controls added 1.5 μL of RNase Free Water.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 4°C for 4 days under light shielding.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. 1 μL of SARS-CoV-2 RNA adjusted to 1250 copies/μL was added to each PCR reaction solution, and RT-PCR was performed as a 50 μL reaction system. Detection targets were two regions in the N gene of SARS-CoV-2, and simultaneous detection was performed for the N1 region using the Cy5 channel and the N2 region using the ROX channel. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 30 cycles at 60°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 sec 95°C 5 sec - 60°C 30 sec 10 cycles (PCR-fluorescence reading)
95°C 5s-60°C 30s 30 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIG. 5 for the N1 region and in FIG. 6 for the N2 region. As a result, in the two target nucleic acid regions targeted for detection, in the negative control, the fluorescence intensity decreased to less than 80% after storage at 4 ° C for 4 days, but the fluorescence intensity was high under the conditions where each antioxidant was added. It was confirmed that the strength was maintained, the storage stability was improved, and the PCR performance was maintained.

Test Example 6.2 Enzyme system 1 step Examination of addition of antioxidant to multiplex RT-PCR reaction solution 2
The reaction solution with the composition shown below is the basic composition, and an antioxidant is added to each reaction solution, stored under each condition, and then in a two-enzyme system 1-step RT-PCR, SARS-CoV- 2 and the target nucleic acid RNA in influenza A were simultaneously detected.
(1) Preparation of Antioxidant Antioxidant was dissolved in water or dimethyl sulfoxide, and the final concentration in the PCR reaction solution during storage was as shown in Table 8 below.

(2-1) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
₅ mM MgSO4
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody 0.1 U/μL MMLV RTase (Toyobo)
After mixing the primer/probe solution described in (2-2) below with the reaction solution, 1.5 μL of each antioxidant was added so that the concentration was as shown in Table 8, and the final volume was 49 μL. An RT-PCR reaction was prepared. Negative controls added 1.5 μL of RNase Free Water.
(2-2) Primer/probe solution Primers and probes for detecting SARS-CoV-2 coronavirus are published by the US 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 the sequences of the N1 and N2 sets (SEQ ID NOS: 12, 15 , 16, 17, 19, 20, 21) were utilized. Influenza virus type A uses the primers and probe sets (SEQ ID NOS: 26, 27, 28, 29, 30) described in the CDC publication "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Primers and Probes" used. TaqMan® probes corresponding to SARS-CoV-2 coronaviruses N1 and N2 (Cy5 channel) and influenza A (ROX channel) were utilized for detection of each amplicon product. Concentrations of probes and primers in the RT-PCR reaction solution were added as described in the same document.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 4°C for 4 days under light shielding.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. To each PCR reaction solution, 1 μL of an RNA solution containing 1250 copies/μL of SARS-CoV-2 and influenza A RNA was added, RT-PCR was performed in a 50 μL reaction system, and two targets were detected simultaneously. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 30 cycles at 60°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 sec 95°C 5 sec - 60°C 30 sec 10 cycles (PCR-fluorescence reading)
95°C 5s-60°C 30s 30 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIGS. As a result, in the negative control, the fluorescence intensity decreased to 79% for SARS-CoV-2 and 78% for influenza A after storage at 4 ° C for 4 days. It maintained a high fluorescence intensity compared to the negative control. As a result, it was confirmed that storage stability was improved and PCR performance was maintained as a multiplex real-time PCR reagent.

Test Example 7.2 Enzyme system 1 step Examination of addition of antioxidant to multiplex RT-PCR reaction solution 3
The reaction solution having the composition shown below is used as a basic composition, an antioxidant is added to this reaction solution, and after storage under the following conditions, in a two-enzyme system 1-step RT-PCR, SARS-CoV- in the reaction solution 2. Three target nucleic acid regions in influenza A and B were simultaneously detected.
(1) Preparation of Antioxidant Sodium sulfite was dissolved in water as an antioxidant, and the final concentration during storage in the PCR reaction solution was as shown in Table 9 below.

(2-1) Preparation of reaction solution and addition of antioxidant Reaction solution (10x buffer diluted to 1x)
(rTaq DNA Polymerase 10xBuffer (Toyobo) accessories)
0.2mM sNTPs Mixture (Toyobo)
₅ mM MgSO4
4.2 ng/μL rTth DNA polymerase (Toyobo)
0.01 μg/μL anti-Tth antibody 0.1 U/μL MMLV RTase (Toyobo)
After mixing the primer/probe solution described in (2-2) below with the reaction solution, 1.5 μL of sodium sulfite was added to the concentration shown in Table 9, and the final volume was 49 μL. A PCR reaction solution was prepared. Negative controls added 1.5 μL of RNase Free Water.
(2-2) Primer/probe solution Primers and probes for detecting SARS-CoV-2 coronavirus are published by the US 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 the sequences of the N1 and N2 sets (SEQ ID NOS: 12, 15 , 16, 17, 19, 20, 21) were utilized. Influenza virus types A and B are the primers and probe sets (SEQ ID NOS: 26, 27, 28, 29) described in the CDC publication "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Primers and Probes" , 30, 31, 32, 33) were utilized. Detection of each amplicon product utilized TaqMan® probes corresponding to SARS-CoV-2 coronaviruses N1 and N2 (Cy5 channel), influenza A (ROX channel) and B (HEX channel). Concentrations of probes and primers in the RT-PCR reaction solution were added as described in the same document.
(3) Storage of PCR reaction solution The prepared PCR reaction solution was stored at 4°C for 4 days under light shielding.
(4) Implementation of PCR Using the PCR reaction solution immediately after preparation and each PCR reaction solution stored under the conditions of (3), PCR was performed in the same manner as described below. To each PCR reaction solution, 1 μL of an RNA solution containing 1250 copies/μL of SARS-CoV-2, influenza A and B RNA was added, RT-PCR was performed in a 50 μL reaction system, and three targets were simultaneously detected. Detected. Real-time PCR reactions were performed using a BioRad CFX96WELL DEEP with the following temperature cycles. Fluorescence readings were taken with an extension step of 30 cycles at 60°C. The relative fluorescence intensity reached, calculated by BioRad's CFX Manager or CFX Maestro software, was then taken as the measured value.
42°C for 5 minutes (reverse transcription conditions)
95°C 10 sec 95°C 5 sec - 60°C 30 sec 10 cycles (PCR-fluorescence reading)
95°C 5s-60°C 30s 30 cycles (PCR-fluorescence reading)
(5) Results The reached relative fluorescence intensity obtained as the measured value at the start was defined as 100%, and compared with the measured values obtained under each condition. The results are shown in FIG. As a result, in the negative control, the fluorescence intensity of SARS-CoV-2, influenza A, and influenza B all decreased after 4 days storage at 4°C, but all of them were high when antioxidants were added. Fluorescence intensity could be maintained. In this example, it was confirmed that the storage stability was improved and the PCR performance was maintained even in the multiplex real-time PCR reagent for amplifying and detecting target nucleic acids at three sites.

INDUSTRIAL APPLICABILITY The present invention is suitably used in molecular biological research, clinical examination, food hygiene management, examination for the prevention of spread of infectious diseases, and the like.

Claims (32)

(I) DNA polymerase, (II) at least one nucleotide selected from the group consisting of oligonucleotides and mononucleotides, (III) divalent or higher metal ions, (IV) buffer, and (V) antioxidant A composition for nucleic acid amplification, comprising: 2. The composition for nucleic acid amplification according to claim 1, comprising (II) a fluorescently labeled probe as a nucleotide. 3. The composition for nucleic acid amplification according to claim 1, further comprising (VI) an anti-DNA polymerase antibody. (I) The composition for nucleic acid amplification according to any one of claims 1 to 3, wherein the DNA polymerase is a DNA polymerase having reverse transcription activity. (I) The composition for nucleic acid amplification according to any one of claims 1 to 4, wherein the DNA polymerase is a DNA polymerase belonging to Family A. 6. The composition for nucleic acid amplification according to any one of claims 1 to 5, further comprising (VII) a reverse transcriptase. (VII) the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMLV)-derived reverse transcriptase, avian myeloblastosis virus (AMV)-derived reverse transcriptase, and variants thereof 7. The composition for nucleic acid amplification according to claim 6, wherein 8. The composition for nucleic acid amplification according to any one of claims 2 to 7, wherein the fluorescently labeled probe is a TaqMan probe. (V) The composition for nucleic acid amplification according to any one of claims 1 to 8, wherein the concentration of antioxidant is 0.001 mM or more. (V) antioxidants such as ascorbic acids, tocopherols, tocotrienols, carotenes, xanthophylls, flavonoids, polyphenols, riboflavins, thiols, gallates, sulfites, alkylphenols, melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sulfur dioxide, and at least one antioxidant selected from the group consisting of trihydroxybutyrophenone (THBP). Item 10. The composition for nucleic acid amplification according to any one of Items 1 to 9. (V) antioxidants such as ascorbic acid, erythorbic acid, tocopherol, tocopherol acetate, tocopherol succinate, tocotrienol, tocotrienol acetate, tocotrienol succinate, retinol, α-carotene, β-carotene, lycopene, lutein, zeaxanthin, canthaxanthin, astaxanthin , rubixanthin, β-cryptoxanthin, capsanthin, fucoxanthin, myxoxanthin, catechin, anthocyanin, tannin, rutin, isoflavone, isoflavane, nobiletin, chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, oleocanthal, oleuropein, resvera Thorol, riboflavin, riboflavin phosphate, lutathione, cysteine, melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sodium sulfite, potassium sulfite, potassium pyrosulfite, gallic acid propyl acid, octyl gallate, dodecyl gallate, dibutylhydroxytoluene, butylhydroxyanisole, butylhydroquinone, sulfur dioxide, trihydroxybutyrophenone, and at least one antioxidant selected from the group consisting of salts thereof; Item 11. The composition for nucleic acid amplification according to any one of items 1 to 10. (III) The composition for nucleic acid amplification according to any one of claims 1 to 11, which contains at least one selected from the group consisting of magnesium ions and manganese ions as the divalent or higher metal ion. 13. The composition for nucleic acid amplification according to any one of claims 1 to 12, which is a premix type reagent for nucleic acid amplification. 14. The composition for nucleic acid amplification according to any one of claims 1 to 13, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 4°C for 4 to 14 days. 15. The composition for nucleic acid amplification according to any one of claims 1 to 14, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 25°C for 4 to 7 days. 16. The composition for nucleic acid amplification according to any one of claims 1 to 15, wherein when left at 25°C for 4 days, the change in color tone measured by any absorbance in the 435 to 490 nm region is suppressed. A composition for nucleic acid amplification comprising (I) a DNA polymerase, (II) at least one nucleotide selected from the group consisting of oligonucleotides and mononucleotides, (III) a divalent or higher metal ion, and (IV) a buffer. and (V) a method for improving the storage stability of the composition for nucleic acid amplification by coexisting an antioxidant. 18. The method according to claim 17, wherein the nucleic acid amplification composition comprises (II) a probe fluorescently labeled as a nucleotide. 19. The method according to claim 17 or 18, wherein the nucleic acid amplification composition further comprises (VI) an anti-DNA polymerase antibody. (I) The method according to any one of claims 17 to 19, wherein the DNA polymerase is a DNA polymerase having reverse transcription activity. (I) The method according to any one of claims 17 to 20, wherein the DNA polymerase is a DNA polymerase belonging to Family A. The method according to any one of claims 17 to 21, wherein the composition for nucleic acid amplification further comprises (VII) reverse transcriptase. (VII) the reverse transcriptase is at least one selected from the group consisting of Moloney murine leukemia virus (MMLV)-derived reverse transcriptase, avian myeloblastosis virus (AMV)-derived reverse transcriptase, and variants thereof 23. The method of claim 22, wherein: The method of any of claims 18-23, wherein the fluorescently labeled probe is a TaqMan probe. 25. The method according to any one of claims 17 to 24, wherein the composition for nucleic acid amplification includes (V) an antioxidant at a concentration of 0.001 mM or higher. (V) antioxidants such as ascorbic acids, tocopherols, tocotrienols, carotenes, xanthophylls, flavonoids, polyphenols, riboflavins, thiols, gallates, sulfites, alkylphenols, melatonin, urobilinogen, At least one antioxidant selected from the group consisting of uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sulfur dioxide, and trihydroxybutyrophenone (THBP) coexists, A method according to any of claims 17-25. (V) antioxidants such as ascorbic acid, erythorbic acid, tocopherol, tocopherol acetate, tocopherol succinate, tocotrienol, tocotrienol acetate, tocotrienol succinate, retinol, α-carotene, β-carotene, lycopene, lutein, zeaxanthin, canthaxanthin, astaxanthin , rubixanthin, β-cryptoxanthin, capsanthin, fucoxanthin, myxoxanthin, catechin, anthocyanin, tannin, rutin, isoflavone, isoflavane, nobiletin, chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, oleocanthal, oleuropein, resvera Thorol, riboflavin, riboflavin phosphate, lutathione, cysteine, melatonin, urobilinogen, uric acid, bilirubin, nordihydroguaiaretic acid, coumarin, catalase, ferritin, albumin, thioredoxin, melanoidin, sodium sulfite, potassium sulfite, potassium pyrosulfite, gallic acid propyl acid, octyl gallate, dodecyl gallate, dibutylhydroxytoluene, butylhydroxyanisole, butylhydroquinone, sulfur dioxide, trihydroxybutyrophenone, and at least one antioxidant selected from the group consisting of salts thereof, A method according to any of claims 17-26. 28. The method according to any one of claims 17 to 27, wherein the composition for nucleic acid amplification contains (III) at least one selected from the group consisting of magnesium ions and manganese ions as the divalent or higher metal ion. The method according to any one of claims 17 to 28, wherein the composition for nucleic acid amplification is a premix type reagent. The method according to any one of claims 17 to 29, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 4°C for 4 to 14 days. The method according to any one of claims 17 to 30, wherein deterioration in nucleic acid amplification reaction performance is suppressed when left at 25°C for 4 to 7 days. 32. The method according to any one of claims 17 to 31, wherein a change in color tone as measured by any absorbance within the 435 to 490 nm region is suppressed when left at 25°C for 4 days.

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