JP7655306B2 - Method for suppressing non-specific nucleic acid amplification - Google Patents

Description

The present invention relates to the generation and amplification of nucleic acid amplification. For example, the present invention relates to compositions and methods useful for the generation and amplification of nucleic acids from ribonucleic acid (RNA) templates, specifically, compositions and methods for the generation and amplification of nucleic acids by reverse transcription using specific nucleic acid polymers. More specifically, the present invention relates to the generation and amplification of nucleic acids by real-time reverse transcription polymerase chain reaction (RT-PCR) in the presence of specific nucleic acid polymers. The method of the present invention enables specific detection of RNA derived from RNA viruses in, for example, fecal samples, blood samples, environmental swabs, etc., and RNA derived from living organisms in biological samples, etc. The present invention can also be used in life science research, clinical diagnosis, food hygiene testing, environmental testing, etc.

Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level where it can be visualized, i.e., hundreds of millions of copies or more. It is widely used not only in the field of life science research, but also in the medical field, such as genetic diagnosis and clinical testing, and in microbial testing of food and the environment. A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR is a method for amplifying a target nucleic acid in a sample by repeating three steps, namely (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) extension of the primer using DNA polymerase, which constitute one cycle. Annealing and extension may be performed in two steps at the same temperature.

When analyzing RNA, reverse transcription (RT) is carried out as a first step in PCR to convert template RNA into cDNA. This is called RT-PCR. RT-PCR can be broadly divided into three types: (1) two-step RT-PCR, in which RT and PCR are carried out discontinuously; (2) one-enzyme one-step RT-PCR, in which RT and PCR are carried out consecutively using a single enzyme; and (3) two-enzyme one-step RT-PCR, in which RT and PCR are carried out consecutively using two types of enzymes, reverse transcriptase and DNA polymerase.

Among RT-PCR methods, one-step RT-PCR is preferred for genetic and viral testing due to its high throughput and the avoidance of contamination caused by opening and closing the reaction vessel during the reaction. Two-enzyme one-step RT-PCR uses at least two types of enzymes: reverse transcriptase and DNA polymerase. On the other hand, one-enzyme one-step RT-PCR uses a DNA polymerase that also has reverse transcription activity, such as Tth DNA polymerase.

The detection sensitivity of one-step RT-PCR is closely related to the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase. To date, in order to improve the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase, the use of amino acid mutants with improved enzyme activity (Patent Document 1) and various additives have been considered (Patent Document 2). In two-enzyme one-step RT-PCR, it is known that the reverse transcriptase itself inhibits PCR, and a method of adding a nucleic acid polymer as a method of reducing inhibition by reverse transcriptase is also known (Patent Document 3). On the other hand, in one-enzyme one-step RT-PCR, PCR is not inhibited by reverse transcriptase.

In addition to the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase, nonspecific gene amplification also affects detection sensitivity. Nonspecific gene amplification refers to the amplification of nucleic acid sequences other than the target gene in PCR. In one-step RT-PCR, all reactions are carried out in the same reaction vessel, so nonspecific amplification causes the depletion of substrates such as deoxynucleotide triphosphate (dNTP) that are required for the target gene amplification that should occur. This prevents the target gene from being amplified in sufficient quantities, leading to a decrease in detection sensitivity.

Many methods have been reported for reducing nonspecific amplification in one-step RT-PCR. For example, optimization of amplification conditions (increasing annealing temperature, reducing the number of cycles, decreasing the amount of enzyme, changing the type of enzyme, decreasing dNTP concentration, decreasing Mg concentration, Mn concentration, decreasing template DNA concentration, etc.), changing primer design, using hot start methods that utilize antibodies or aptamers, and using modified oligonucleotides (such as PNA and LNA) that are believed to increase specificity are known.

However, although such methods can be effective in suppressing non-specific amplification, they are often insufficiently effective or end up suppressing specific amplification as well. Furthermore, they often require consideration of RT-PCR conditions and primer design, which can be time-consuming. Thus, there has been a demand for the development of a simpler method that can suppress non-specific amplification in one-step RT-PCR.

International Publication No. 2018096961A JP 2018-000138 A Patent No. 4777497

FIG. 1 shows the results (reference values) of the base length distribution of potassium polyinosinate used in Test Examples 1, 2, 6, and 7, measured using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation). FIG. 1 shows the base length distribution of potassium polycytidylate used in Test Example 3, as measured using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation) (reference values). FIG. 1 shows the results (reference values) of the base length distribution of potassium polyadenylate used in Test Example 3, measured using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation). FIG. 1 shows the results (reference values) of the base length distribution of potassium polyguanylate used in Test Example 4, measured using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation). FIG. 13 is a diagram showing the results (reference values) of the base length distribution of polyinosinic acid-polycytidylic acid sodium salt (Poly(I:C)) used in Test Example 5, measured using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation). 1 is a graph showing the results of detection of norovirus G1 gene when potassium polyinosinate was added in Test Example 1. 1 is a graph showing the results of detection of norovirus G2 gene when potassium polyinosinate was added in Test Example 1. 1 is a graph showing the results of detection of norovirus G1 gene when potassium polyinosinate was added at a high concentration in Test Example 2. 1 is a graph showing the results of detection of norovirus G2 genes when potassium polyinosinate was added at a high concentration in Test Example 2. 1 is a graph showing the results of detection of the norovirus G1 gene when potassium polyadenylate was added in Test Example 3. 1 is a graph showing the results of detection of norovirus G2 gene when potassium polyadenylate was added in Test Example 3. 1 is a graph showing the results of detection of the norovirus G1 gene when potassium polycytidylate was added in Test Example 3. 1 is a graph showing the results of detection of norovirus G2 gene when potassium polycytidylate was added in Test Example 3. 1 is a graph showing the results of detection of the norovirus G1 gene when potassium polyguanylate and polydeoxythymidylic acid were added in Test Example 4. 1 is a graph showing the results of detection of norovirus G2 gene when potassium polyguanylate and polydeoxythymidylic acid were added in Test Example 4. 1 is a graph showing the results of detection of norovirus G1 gene when polyinosinic acid-sodium polycytidylate (Poly(I:C)) was added in Test Example 5. 1 is a graph showing the results of detection of norovirus G2 gene when polyinosinic acid-sodium polycytidylate (Poly(I:C)) was added in Test Example 5. 1 is a graph showing the detection results of region 1 of the SARS-CoV-2 N gene when potassium polyinosinate was added in Test Example 6. 1 is a graph showing the detection results of region 2 of the SARS-CoV-2 N gene when potassium polyinosinate was added in Test Example 6. 1 is a graph showing the results of influenza A gene detection when potassium polyinosinate was added in Test Example 7. 1 is a graph showing the results of influenza B gene detection when potassium polyinosinate was added in Test Example 7.

The present invention has been made in consideration of the above-mentioned conventional technology, and aims to provide a further useful means that is a simple method yet can effectively suppress non-specific nucleic acid amplification when amplifying nucleic acid from template RNA by one-step RT-PCR.

In view of the above circumstances, the inventors conducted intensive research and discovered that the use of a specific nucleic acid polymer can suppress non-specific nucleic acid amplification in one-step RT-PCR, enabling specific detection of target nucleic acid with high sensitivity, thereby completing the present invention.

Representative aspects of the present invention are as follows:
[Item 1] A method for amplifying nucleic acid from template RNA by one-step RT-PCR, comprising suppressing non-specific nucleic acid amplification using at least one type of nucleic acid polymer selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers.
[Item 2] The method according to Item 1, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polythymidylic acid, polyuridylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, polydeoxythymidylic acid, polydeoxyuridylic acid, and salts thereof.
[Item 3] The method according to Item 1 or 2, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, and salts thereof.
[Item 4] The method according to any one of Items 1 to 3, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof.
[Item 5] The method according to any one of Items 1 to 4, wherein the nucleic acid polymer comprises a nucleic acid polymer having a total length of 30 to 10,000 bases.
[Item 6] The method according to any one of Items 1 to 5, wherein the nucleic acid polymer is allowed to coexist in a one-step RT-PCR reaction solution at a final concentration of 0.1 ng/μL or more and 1000 ng/μL or less.
[Item 7] The method according to any one of Items 1 to 5, wherein the one-step RT-PCR is a one-enzyme type one-step RT-PCR.
[Item 8] The method according to any one of Items 1 to 7, wherein the DNA polymerase used in the one-step RT-PCR is a thermostable DNA polymerase having reverse transcription activity belonging to family A.
[Item 9] The method according to any one of Items 1 to 8, wherein the thermostable DNA polymerase having reverse transcription activity belonging to Family A is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof.
[Item 10] The method according to Item 9, wherein the mutant has an amino acid sequence that is 90% or more identical to the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2) and exhibits reverse transcription activity and thermostable DNA polymerase activity.
[Item 11] The method according to Item 9 or 10, wherein the mutant has an amino acid sequence having one or several amino acid deletions, substitutions and/or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2), and exhibits reverse transcription activity and thermostable DNA polymerase activity.
[Item 12] The method according to any one of Items 1 to 11, wherein the template RNA is RNA derived from a biological sample, an environmental sample, or a cell.
[Item 13] The method according to Item 12, wherein the biological sample is at least one selected from the group consisting of feces, urine, vomit, saliva, sputum, throat swab, nasal swab, nasal mucus, blood, plasma, and serum.
[Item 14] The method according to Item 12, wherein the environmental sample is at least one selected from the group consisting of a swab test sample, a soil sample, and a sewage sample.
[Item 15] A composition for use in a one-step RT-PCR reaction in which non-specific nucleic acid amplification is suppressed, comprising at least one type of nucleic acid polymer selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers.
[Item 16] The composition according to Item 15, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polythymidylic acid, polyuridylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, polydeoxythymidylic acid, polydeoxyuridylic acid, and salts thereof.
[Item 17] The composition according to Item 15 or 16, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, and salts thereof.
[Item 18] The composition according to any one of Items 15 to 17, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof.
[Item 19] The method according to Item 1 or the composition according to Item 15, wherein the nucleic acid polymer contains 60 mol% or more of nucleotide-derived structural units selected from the group consisting of inosinic acid, cytidylic acid, guanylic acid, deoxyinosinic acid, deoxycytidylic acid, deoxyguanylic acid, derivatives thereof, and salts thereof, out of all structural units.
[Item 20] The method according to item 1 or 19, or the composition according to item 15 or 19, wherein the nucleic acid polymer contains 90 mol% or more of nucleotide-derived structural units selected from the group consisting of inosinic acid, cytidylic acid, guanylic acid, deoxyinosinic acid, deoxycytidylic acid, deoxyguanylic acid, derivatives thereof, and salts thereof, out of all structural units.
[Item 21] The method according to Item 1, 19 or 20, or the composition according to Item 15, 19 or 20, wherein the nucleic acid polymer contains 60 mol% or more of nucleotide-derived structural units selected from the group consisting of inosinic acid, deoxyinosinic acid, derivatives thereof, and salts thereof among all structural units.
[Item 22] The method according to any one of Items 1, 19 to 21, or the composition according to any one of Items 15, 19 to 21, wherein the nucleic acid polymer contains 90 mol % or more of nucleotide-derived structural units selected from the group consisting of inosinic acid, deoxyinosinic acid, derivatives thereof, and salts thereof among all structural units.
[Item 23] The composition according to any one of Items 15 to 22, wherein the nucleic acid polymer comprises a nucleic acid polymer having a total length of 30 to 10,000 bases.
[Item 24] The composition according to any one of Items 15 to 23, wherein the one-step RT-PCR is a one-enzyme type one-step RT-PCR.
[Item 25] The composition according to any one of Items 15 to 24, which is prepared so that the final concentration of the nucleic acid polymer in a one-step RT-PCR reaction solution is 0.1 ng/μL or more and 1000 ng/μL or less.
[Item 26] The composition according to any one of Items 15 to 25, further comprising a thermostable DNA polymerase having reverse transcription activity belonging to Family A.
[Item 27] The composition according to Item 26, wherein the thermostable DNA polymerase having reverse transcription activity belonging to Family A is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof.
[Item 28] The composition described in Item 27, wherein the mutant has an amino acid sequence that is 90% or more identical to the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2) and exhibits reverse transcription activity and thermostable DNA polymerase activity.
[Item 29] The composition described in Item 27 or 28, wherein the mutant has an amino acid sequence having one or several amino acid deletions, substitutions and/or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2), and exhibits reverse transcription activity and thermostable DNA polymerase activity. [Item 30] The composition described in any of Items 15 to 29, which is used for nucleic acid amplification from RNA derived from a biological sample, an environmental sample or a cell.
[Item 31] The composition described in Item 30, wherein the biological sample is at least one selected from the group consisting of feces, urine, vomit, saliva, sputum, throat swabs, nasal swabs, nasal mucus, blood, plasma, and serum.
[Item 32] The composition according to Item 30, wherein the environmental sample is at least one selected from the group consisting of a swab test sample, a soil sample, and a sewage sample.
[Item 33] A kit for use in a one-step RT-PCR reaction in which non-specific nucleic acid amplification is suppressed, comprising the composition according to any one of Items 15 to 32.

According to the present invention, by having a specific nucleic acid polymer coexist in the reaction solution, non-specific nucleic acid amplification in a one-step RT-PCR reaction can be suppressed, thereby increasing the amount of specific amplification product produced from the target nucleic acid intended to be amplified, which may improve detection sensitivity.

The present invention will be described in more detail below with reference to the embodiments of the present invention, but the present invention is not limited thereto. It should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified.
In addition, all non-patent and patent documents described in this specification are incorporated herein by reference. In this specification, "~" means "more than or equal to, less than or equal to", for example, if the specification states "X~Y", it means "more than or equal to X, less than or equal to Y". In addition, in this specification, "and/or" means either one or both. In addition, in this specification, the expression in the singular form should be understood to include the concept of the plural form, unless otherwise specified.

One embodiment of the present invention is a method for amplifying nucleic acids by a one-step RT-PCR reaction using RNA contained in a sample as a template, in which non-specific nucleic acid amplification is suppressed by carrying out the one-step RT-PCR reaction in the coexistence of a specific nucleic acid polymer. Here, non-specific nucleic acid amplification means that a nucleic acid sequence other than the target sequence of the template RNA is synthesized in the one-step RT-PCR.

In general, when amplifying a nucleic acid (e.g., a gene) using RNA as a template, a reverse transcription reaction (also called an RT reaction) is first performed to convert the template RNA into cDNA, and the cDNA obtained is amplified by a PCR reaction. One-step RT-PCR refers to a method of amplifying a nucleic acid from a template RNA by performing the above-mentioned RT reaction and PCR reaction consecutively or in parallel. Known examples of one-step RT-PCR include a two-enzyme one-step RT-PCR reaction in which the RT reaction and the PCR reaction are performed consecutively using two different enzymes (reverse transcriptase and DNA polymerase), and a single-enzyme one-step RT-PCR in which the RT reaction and the PCR reaction are performed consecutively or in parallel using a single enzyme (a DNA polymerase that also has reverse transcriptase activity, such as Tth DNA polymerase). The present invention can be performed with any one-step RT-PCR, but it is preferable to perform it with a one-enzyme one-step RT-PCR from the viewpoint of more reliably suppressing nonspecific nucleic acid amplification and making it easier to detect the target nucleic acid with high sensitivity.

In one-step RT-PCR, all reactions are preferably carried out in the same reaction vessel. When one-step RT-PCR is carried out in the same reaction vessel in this way, if non-specific nucleic acid amplification occurs, substrates such as deoxynucleotide triphosphate (dNTP) that are necessary for the target nucleic acid amplification that should occur may be depleted, and the target nucleic acid may not be amplified in sufficient amounts, resulting in a decrease in detection sensitivity. Therefore, the method of the present invention, which can suppress non-specific nucleic acid amplification in one-step RT-PCR reactions, can suppress a decrease in the detection sensitivity of such target nucleic acids. Therefore, the method of the present invention can also be said to be, for example, a method for increasing the production amount of specific nucleic acid amplification products, a method for improving the specific amplification of target nucleic acids, etc.

The nucleic acid polymer used in the present invention is preferably at least one selected from the group consisting of inosinic acid polymer, cytidylic acid polymer, guanylic acid polymer, adenylic acid polymer, thymidylic acid polymer, uridylic acid polymer, deoxyinosinic acid polymer, deoxycytidylic acid polymer, deoxyguanylic acid polymer, deoxyadenylic acid polymer, deoxythymidylic acid polymer, and deoxyuridylic acid polymer. Among them, from the viewpoint of more effectively suppressing non-specific nucleic acid amplification in one-step RT-PCR, at least one selected from the group consisting of inosinic acid polymer, cytidylic acid polymer, guanylic acid polymer, deoxyinosinic acid polymer, deoxycytidylic acid polymer, and deoxyguanylic acid polymer is more preferable, and inosinic acid polymer and/or deoxyinosinic acid polymer are particularly preferable. In the present invention, two or more kinds of the above-mentioned nucleic acid polymers may be used, for example, an inosinic acid polymer and a cytidylic acid polymer may be used in combination, or a deoxyinosinic acid polymer and a cytidylic acid polymer may be used in combination. When two or more types of nucleic acid polymers are used, one of the nucleic acid polymers is preferably an inosinic acid polymer or a deoxyinosinic acid polymer.
Each polymer will be described in detail below.

(1) Inosinic acid polymer The term "inosinic acid polymer" is used to include inosinic acid homopolymer (polyinosinic acid), inosinic acid copolymer, their derivatives, and their salts. In addition, a double-stranded nucleic acid polymer called poly(I:C) or the like, which is an annealed inosinic acid homopolymer (polyinosinic acid) and cytidylic acid homopolymer (polycytidylic acid), is known in the art. In the present invention, like the above double-stranded nucleic acid polymer, a double-stranded nucleic acid polymer in which at least one of the strands is an inosinic acid polymer (e.g., polyinosinic acid) is also included in the inosinic acid polymer.
An inosinic acid copolymer is a polymer having building blocks derived from inosinic acid (IMP) and building blocks derived from nucleotides other than IMP.
A nucleotide-derived building block, such as an IMP, refers to a unit made up of nucleotides, such as those represented by the following formula:

(In the above formula, R is a hydrogen atom or hydroxyl, and Base is a nucleic acid base such as inosine, cytosine, guanine, adenine, thymine, or uracil.)
Nucleotides other than IMP are not particularly limited, and may be ribonucleotides or deoxyribonucleotides. Examples of nucleotides other than IMP include deoxyinosinic acid (dIMP), cytidylic acid (CMP), deoxycytidylic acid (dCMP), guanylic acid (GMP), deoxyguanylic acid (dGMP), adenylic acid (AMP), deoxyadenylic acid (dAMP), thymidylic acid (TMP), deoxythymidylic acid (dTMP), uridylic acid (UMP), deoxyuridylic acid (dUMP), and combinations of two or more of these. Among these, at least one selected from the group consisting of dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and dIMP is more preferred.
The above-mentioned "derivative" refers to a polymer in which the basic skeleton of the original polymer is maintained while a part of the structure is modified. Modifications include, for example, oxidation, reduction, atomic substitution, introduction of functional groups, and the like. The above-mentioned "derivative" is preferably a polymer in which a functional group (e.g., a fluorine atom, a bromine atom, an iodine atom, a lower alkyl group (e.g., a C1-6 alkyl group such as a methyl group, an ethyl group, etc.), an amino group, a mercapto group, or a combination of two or more of these) is introduced into the base moiety in at least a part of the structural units of the original polymer, and/or a phosphate moiety is replaced with a thiophosphate moiety.
As the above-mentioned "salt", an alkali metal salt is preferable, and a sodium salt or a potassium salt is more preferable.
In the inosinic acid polymer, the IMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of all constitutional units. In addition, the IMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of all constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the inosinic acid polymer, the number of repeats of the IMP-derived constitutional unit (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more, 150 or more. The number of repeats of the IMP-derived constitutional unit may be, for example, 10,000 or less, 8,000 or less, and may be, for example, 5,000 or less, 3,000 or less, 1,000 or less, 800 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(2) Cytidylic acid polymer The term "cytidylic acid polymer" is used to include cytidylic acid homopolymer (polycytidylic acid), cytidylic acid copolymer, their derivatives, and their salts. In addition, a double-stranded nucleic acid polymer called poly(I:C) or the like, which is an annealed inosinic acid homopolymer (polyinosinic acid) and cytidylic acid homopolymer (polycytidylic acid), is known in the art. In the present invention, like the above double-stranded nucleic acid polymer, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a cytidylic acid polymer (e.g., polycytidylic acid) is also included in the cytidylic acid polymer.
Cytidylic acid copolymer is a polymer having a constitutional unit derived from CMP and a constitutional unit derived from a nucleotide other than CMP. The nucleotide other than CMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than CMP include IMP, dIMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the cytidylic acid polymer, the CMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The CMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the cytidylic acid polymer, the number of repeats of the CMP-derived constitutional unit (which may also be expressed as the base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the CMP-derived constitutional unit may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, 1,000 or less, 800 or less, or 500 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(3) Guanylic Acid Polymer The term "guanylic acid polymer" is used to mean a guanylic acid homopolymer (polyguanylic acid), a guanylic acid copolymer, a derivative thereof, and a salt thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a guanylic acid polymer (e.g., polyguanylic acid) is also included in the guanylic acid polymer.
The guanylic acid copolymer is a polymer having a constitutional unit derived from GMP and a constitutional unit derived from a nucleotide other than GMP. The nucleotide other than GMP is not particularly limited and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than GMP include IMP, dIMP, CMP, dCMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the guanylic acid polymer, the GMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The GMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the guanylic acid polymer, the number of repeats of the GMP-derived constitutional unit (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the GMP-derived constitutional unit may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, 1,000 or less, or 600 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(4) Adenylic Acid Polymer The term "adenylic acid polymer" is used to mean an adenylic acid homopolymer (polyadenylic acid), an adenylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, the term "adenylic acid polymer" also includes a double-stranded nucleic acid polymer in which at least one strand of the double strand is an adenylic acid polymer (e.g., polyadenylic acid).
Adenylic acid copolymer is a polymer having a constitutional unit derived from AMP and a constitutional unit derived from a nucleotide other than AMP. The nucleotide other than AMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than AMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the adenylic acid polymer, the AMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of all the constitutional units. In addition, the AMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of all the constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the adenylic acid polymer, the number of repeats of the AMP-derived constitutional unit (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the AMP-derived constitutional unit may be, for example, 10,000 or less, 8,000 or less, or 5,000 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(5) Thymidylic Acid Polymer The term "thymidylic acid polymer" is used to mean a thymidylic acid homopolymer (polythymidylic acid), a thymidylic acid copolymer, a derivative thereof, and a salt thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a thymidylic acid polymer (e.g., polythymidylic acid) is also included in the thymidylic acid polymer.
Thymidylic acid copolymer is a polymer having a constitutional unit derived from TMP and a constitutional unit derived from a nucleotide other than TMP. The nucleotide other than TMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than TMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the thymidylic acid polymer, the TMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The TMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the thymidylic acid polymer, the number of repeats of the constitutional unit derived from TMP (which may also be expressed as base length, etc.) may be, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more. The number of repeats of the constitutional unit derived from TMP may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, 1,000 or less, and may be, for example, less than 100, 95 or less, or 90 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(6) Uridylic Acid Polymer The term "uridylic acid polymer" is used to include uridylic acid homopolymer (polyuridylic acid), uridylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, the term "uridylic acid polymer" also includes a double-stranded nucleic acid polymer in which at least one strand of the double strand is a uridylic acid polymer (e.g., polyuridylic acid).
The uridylic acid copolymer is a polymer having a constitutional unit derived from UMP and a constitutional unit derived from a nucleotide other than UMP. The nucleotide other than UMP is not particularly limited and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than UMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the uridylic acid polymer, the UMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of all constitutional units. The UMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of all constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the uridylic acid polymer, the number of repeats of the constitutional unit derived from UMP (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more, 150 or more. The number of repeats of the constitutional unit derived from UMP may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(7) Deoxyinosinic Acid Polymer The term "deoxyinosinic acid polymer" is used to mean a deoxyinosinic acid homopolymer (polydeoxyinosinic acid), a deoxyinosinic acid copolymer, a derivative thereof, and a salt thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxyinosinic acid polymer (e.g., polydeoxyinosinic acid) is also included in the deoxyinosinic acid polymer.
Deoxyinosinic acid copolymer is a polymer having a constitutional unit derived from dIMP and a constitutional unit derived from a nucleotide other than dIMP. The nucleotide other than dIMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dIMP include IMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, CMP, dCMP, GMP, and dGMP is more preferred, and IMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxyinosinic acid polymer, the dIMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The dIMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxyinosinic acid polymer, the number of repeats of the constitutional unit derived from dIMP (which may also be expressed as the base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more, 150 or more. The number of repeats of the constitutional unit derived from dIMP may be, for example, 10,000 or less, 8,000 or less, and may be, for example, 5,000 or less, 3,000 or less, 1,000 or less, 800 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(8) Deoxycytidylic Acid Polymer The term "deoxycytidylic acid polymer" is used to mean a deoxycytidylic acid homopolymer (polydeoxycytidylic acid), a deoxycytidylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxycytidylic acid polymer (e.g., polydeoxycytidylic acid) is also included in the deoxycytidylic acid polymer.
Deoxycytidylic acid copolymer is a polymer having a constitutional unit derived from dCMP and a constitutional unit derived from a nucleotide other than dCMP. The nucleotide other than dCMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dCMP include IMP, dIMP, CMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxycytidylic acid polymer, the constituent units derived from dCMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constituent units. The constituent units derived from dCMP may be 100 mol% (i.e., homopolymer) of the total constituent units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxycytidylic acid polymer, the number of repeats of the constitutional unit derived from dCMP (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the constitutional unit derived from dCMP may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, 1,000 or less, 800 or less, or 500 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(9) Deoxyguanylic Acid Polymer The term "deoxyguanylic acid polymer" is used to mean a deoxyguanylic acid homopolymer (polydeoxyguanylic acid), a deoxyguanylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxyguanylic acid polymer (e.g., polydeoxyguanylic acid) is also included in the deoxyguanylic acid polymer.
Deoxyguanylic acid copolymer is a polymer having a constitutional unit derived from dGMP and a constitutional unit derived from a nucleotide other than dGMP. The nucleotide other than dGMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dGMP include IMP, dIMP, CMP, dCMP, GMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, and GMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxyguanylic acid polymer, the constitutional unit derived from dGMP is, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. In addition, the constitutional unit derived from dGMP may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxyguanylic acid polymer, the number of repeats of the constitutional unit derived from dGMP (which may also be expressed as the base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the constitutional unit derived from dGMP is, for example, 10,000 or less, 8,000 or less, preferably 6,500 or less, more preferably 6,000 or less, and may be 5,000 or less, 3,000 or less, 1,000 or less, or 600 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(10) Deoxyadenylic Acid Polymer The term "deoxyadenylic acid polymer" is used to mean a deoxyadenylic acid homopolymer (polydeoxyadenylic acid), a deoxyadenylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxyadenylic acid polymer (e.g., polydeoxyadenylic acid) is also included in the deoxyadenylic acid polymer.
Deoxyadenylic acid copolymer is a polymer having a constitutional unit derived from dAMP and a constitutional unit derived from a nucleotide other than dAMP. The nucleotide other than dAMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dAMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, and AMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxyadenylic acid polymer, the dAMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The dAMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxyadenylic acid polymer, the number of repeats of the constitutional unit derived from dAMP (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, even more preferably 60 or more, and even more preferably 70 or more, and may be, for example, 100 or more. The number of repeats of the constitutional unit derived from dAMP may be, for example, 10,000 or less, 8,000 or less, or 5,000 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(11) Deoxythymidylic Acid Polymer The term "deoxythymidylic acid polymer" is used to mean a deoxythymidylic acid homopolymer (polydeoxythymidylic acid), a deoxythymidylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxythymidylic acid polymer (e.g., polydeoxythymidylic acid) is also included in the deoxythymidylic acid polymer.
Deoxythymidylic acid copolymer is a polymer having a constitutional unit derived from dTMP and a constitutional unit derived from a nucleotide other than dTMP. The nucleotide other than dTMP is not particularly limited, and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dTMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, UMP, dUMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxythymidylic acid polymer, the dTMP-derived constitutional units are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of the total constitutional units. The dTMP-derived constitutional units may be 100 mol% (i.e., homopolymer) of the total constitutional units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxythymidylic acid polymer, the number of repeats of the constitutional unit derived from dTMP (which may also be expressed as the base length, etc.) may be, for example, 30 or more, preferably 40 or more, more preferably 50 or more, preferably 60 or more, more preferably 70 or more. The number of repeats of the constitutional unit derived from dTMP may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

(12) Deoxyuridylic Acid Polymer The term "deoxyuridylic acid polymer" is used to mean a deoxyuridylic acid homopolymer (polydeoxyuridylic acid), a deoxyuridylic acid copolymer, derivatives thereof, and salts thereof. In the present invention, a double-stranded nucleic acid polymer in which at least one strand of the double strand is a deoxyuridylic acid polymer (e.g., polydeoxyuridylic acid) is also included in the deoxyuridylic acid polymer.
Deoxyuridylic acid copolymer is a polymer having a constitutional unit derived from dUMP and a constitutional unit derived from a nucleotide other than dUMP. The nucleotide other than dUMP is not particularly limited and may be a ribonucleotide or a deoxyribonucleotide. Examples of the nucleotide other than dUMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, and combinations of two or more of these. Among these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferred, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP is more preferred, and at least one selected from the group consisting of IMP and dIMP is more preferred.
The above "derivatives" and "salts" include those similar to those described above in "(1) Inosinic acid polymer."
In the deoxyuridylic acid polymer, the constituent units derived from dUMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of all the constituent units. The constituent units derived from dUMP may be 100 mol% (i.e., homopolymer) of all the constituent units, or may be, for example, 99.9 mol% or less, or 99 mol% or less.
In the deoxyuridylic acid polymer, the number of repeats of the constitutional unit derived from dUMP (which may also be expressed as base length, etc.) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, preferably 60 or more, more preferably 70 or more, and may be, for example, 100 or more, 150 or more. The number of repeats of the constitutional unit derived from dUMP may be, for example, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less. The repeats may be continuous or discontinuous, and are preferably continuous.

In a preferred embodiment, the nucleic acid polymer is a nucleic acid polymer containing, for example, 60 mol% or more, preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of a nucleotide-derived structural unit selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, their derivatives, and their salts in all structural units. The upper limit is not particularly limited, and for example, these structural units may be 100 mol%, 99.9 mol% or less, or 99 mol% or less of the total structural units of the nucleic acid polymer.
In a more preferred embodiment, the nucleic acid polymer contains, for example, 60 mol% or more, preferably 65 mol% or more, more preferably 70 mol% or more, more preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, more preferably 95 mol% or more of a nucleotide-derived structural unit selected from the group consisting of IMP, dIMP, derivatives thereof, and salts thereof in all structural units. The upper limit is not particularly limited, and for example, these structural units may be 100 mol% or less, or 99.9 mol% or less of the total structural units of the nucleic acid polymer.

In a preferred embodiment, the nucleic acid polymer is at least one selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polythymidylic acid, polyuridylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, polydeoxythymidylic acid, polydeoxyuridylic acid, and salts thereof.
In a more preferred embodiment, the nucleic acid polymer is at least one selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, and salts thereof.
In a more preferred embodiment, the nucleic acid polymer is at least one selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, and salts thereof.
In a more preferred embodiment, the nucleic acid polymer is at least one selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof.

The nucleic acid polymer preferably includes the nucleic acid polymer having a total length of 25 to 15,000 bases, preferably the nucleic acid polymer having a total length of 30 to 10,000 bases, more preferably the nucleic acid polymer having a total length of 40 to 9,000 bases, and more preferably the nucleic acid polymer having a total length of 50 to 8,000 bases, and may be, for example, 100 to 5,000 bases. For example, the content of the nucleic acid polymer having a total length of 25 to 15,000 bases, 30 to 10,000 bases, 40 to 9,000 bases, 50 to 8,000 bases, or 100 to 5,000 bases is, for example, 70 mol% or more, preferably 80 mol% or more, more preferably 85 mol% or more, more preferably 90 mol% or more, and more preferably 95 mol% or more, relative to the entire nucleic acid polymer.
That is, the nucleic acid polymer may be a mixture of nucleic acid polymers of various base lengths, for example, a mixture of nucleic acid polymers substantially distributed in the range of 25 to 15,000 base lengths, preferably a mixture of nucleic acid polymers substantially distributed in the range of 30 to 10,000 base lengths, more preferably a mixture of nucleic acid polymers substantially distributed in the range of 40 to 9,000 base lengths, and even more preferably a mixture of nucleic acid polymers substantially distributed in the range of 50 to 8,000 base lengths, and for example, a mixture of nucleic acid polymers substantially distributed in the range of 100 to 5,000 base lengths can also be used. "Substantially distributed in the range of base lengths A to B" means that nucleic acid polymers of base lengths A to B account for, for example, 70 mol % or more of the entire nucleic acid polymer.
The nucleic acid polymer may be, for example, a mixture of nucleic acid polymers exhibiting a distribution having a peak (or average value or mode) in the vicinity of 30 to 3000 base lengths, a mixture of nucleic acid polymers exhibiting a distribution having a peak (or average value or mode) in the vicinity of 50 to 1500 base lengths, or preferably a mixture of nucleic acid polymers exhibiting a distribution having a peak (or average value or mode) in the vicinity of 60 to 1000 base lengths, or a mixture of nucleic acid polymers exhibiting a distribution having a peak (or average value or mode) in the vicinity of 70 to 800 base lengths may also be used. "Around" means that the lower limit and/or upper limit may be increased or decreased by 10%.
The total length of the nucleic acid polymer is, for example, 30 bases or more, preferably 40 bases or more, more preferably 50 bases or more, even more preferably 60 bases or more, and even more preferably 70 bases or more, and may be, for example, 100 bases or more, 150 bases or more. Thus, the total length of the nucleic acid polymer is preferably longer than the base length designed as a probe or primer. The total length of the nucleic acid polymer may be, for example, 10,000 bases or less, or even 8,000 bases or less, or may be, for example, 5,000 bases or less.

The nucleic acid polymer can be synthesized by any method that can be used to synthesize oligonucleotides, such as the phosphate triester method, the H-phosphonate method, the thiophosphonate method, etc. Natural products may also be used, and commercially available products can also be used. Examples of commercially available nucleic acid polymers that can be used include potassium polyinosinate (Sigma), potassium polyadenylate (Sigma), and potassium polyinosinate (Santa Cruz).

In one embodiment, the one-step RT-PCR reaction solution used in the present invention contains, for example, the above-mentioned nucleic acid polymer, template RNA, primers, deoxyribonucleotides, and a DNA polymerase having reverse transcription activity. It may also contain other components such as reverse transcriptase, etc., as desired. Therefore, the composition for use in the one-step RT-PCR reaction of the present invention may also contain the above-mentioned components as desired.
In the RT-PCR reaction solution, the concentration of the nucleic acid polymer is, for example, 0.1 ng/μL or more, more preferably 1 ng/μL or more, and even more preferably 10 ng/μL or more, and may be, for example, 50 ng/μL or more, 100 ng/μL or more, 200 ng/μL or more, or 500 ng/μL or more, from the viewpoint of more effectively suppressing nonspecific nucleic acid amplification in a one-step RT-PCR reaction. The concentration of the nucleic acid polymer may be, for example, 100,000 ng/μL or less, preferably 5000 ng/μL or less, more preferably 3000 ng/μL or less, 2000 ng/μL or less, 1500 ng/μL or less, or 1000 ng/μL or less, and may be, for example, 800 ng/μL or less, or 500 ng/μL or less. Therefore, the composition to be used in the one-step RT-PCR reaction of the present invention preferably contains the nucleic acid polymer in an amount adjusted so that the final concentration in the one-step RT-PCR reaction solution is the above-mentioned concentration.

The template RNA may be RNA derived from a biological sample such as tissue, body fluid, or secretion collected from a living organism (e.g., biological tissue, body fluid, or secretion that may contain a pathogenic microorganism such as a virus), an environmental sample, or a cell (e.g., a cultured cell, etc.). For example, it may be RNA extracted from a biological sample, an environmental sample, or a cell, or it may be RNA that has been further purified after extraction processing (e.g., purified RNA that has been treated by any purification means known in the art, such as ethanol precipitation, column purification, etc.), and may be any RNA.
In one embodiment, when RNA is extracted from tissue, the location of the tissue is not particularly limited and may be any tissue. A method for extracting RNA from tissue usually includes a step of isolating cells using a cell strainer or trypsin treatment, but the method is not particularly limited.
In another embodiment, when RNA is extracted from cells, there is no particular limitation, and any type of cell may be used. The number of cells can be adjusted appropriately using a cell sorter. For example, RNA extracted from a small number of cells (e.g., 1 to 100 cells, preferably 1 to 10 cells, more preferably 1 or 2 cells, more preferably 1 cell) can also be used. The method for extracting RNA from cells usually includes a step of lysing cells using a cell lysis composition containing a cell lysis agent. Examples of the cell lysis agent include surfactants and chaotropic agents. Examples of the surfactant include anionic surfactants (e.g., sodium dodecyl sulfate, sodium cholate, sodium deoxycholate), cationic surfactants (e.g., cetyltrimethylammonium bromide), nonionic surfactants (e.g., octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate), and zwitterionic surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). Examples of chaotropic agents include urea and lithium salts such as lithium perchlorate. The cell lysis agent is usually dissolved in water (preferably nuclease-free water) and used in the form of an aqueous solution. The cell lysis composition may contain a protease (e.g., protease K), an RNase inhibitor, or a combination of two or more of these.
The cell lysis composition may or may not contain the above-mentioned nucleic acid polymer. When RNA extracted from cells using a cell lysis composition containing the above-mentioned nucleic acid polymer is used as template RNA, the composition for use in the one-step RT-PCR reaction does not need to contain the above-mentioned nucleic acid polymer separately.

In another embodiment, when RNA is extracted from a biological sample, examples of the biological sample include feces (excreted feces, rectal feces), urine, vomit, saliva, sputum, pharyngeal swabs, nasal swabs, nasal mucus, blood (whole blood), plasma, serum, etc., but are not limited thereto, and can be used for anything derived from a living organism, and can be appropriately selected according to the purpose. In a preferred embodiment, a biological sample such as a fecal sample, a blood sample, or a pharyngeal swabs sample is used as the biological sample. The target template RNA may be RNA derived from a living organism, or may be RNA derived from a microorganism or virus contained in the biological sample. In addition, the sample may be directly subjected to detection, or may be a sample obtained by suspending the sample in water, physiological saline, or a buffer solution in order to reduce the influence of impurities on the reaction and obtain more stable test results. The buffer solution is not particularly limited, but examples include Hank's buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and Tricine buffer solution.

In yet another embodiment, the present invention can be implemented to amplify nucleic acids from RNA in environmental samples such as swab test samples. For example, the present invention can be used to detect target RNA from swab test samples, soil samples, sewage samples, etc. as described above, but is not particularly limited, and any sample derived from the environment can be used. Swab tests are useful for clarifying the route of contamination by viruses and bacteria and understanding the contamination status of facility environments, etc. In the present invention, swab tests are not particularly limited, but are, for example, samples obtained by wiping the relevant area or equipment with a cotton swab, eluting in water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitation, etc. Specific swab test procedures include "Improvement of Norovirus Testing Method for Swab Samples" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html), but are not particularly limited, and methods similar to this are widely included. Examples of places to be wiped include cooking utensils such as cutting boards, knives, dishcloths, and tableware, refrigerator handles, toilet and bathroom doorknobs, faucets in the washroom, kitchen, toilet, and bathroom, the hands and fingers of cooks, bathrooms, toilets, washbasins, handrails, living rooms, etc. In addition, although it is not a wipe test, it can also be applied to concentrated sewage samples as an environmental test.

When the test subject contained in the biological sample is an RNA virus, there is no particular limitation, and it may be an RNA virus having an envelope derived from a lipid bilayer membrane, or a non-enveloped RNA virus.
Examples of enveloped RNA viruses include Flaviviridae viruses (e.g., Hepatitis C virus, Japanese encephalitis virus, Zika virus); Togaviridae viruses (e.g., Rubella virus, Chikungunya virus); Coronaviridae viruses (e.g., SARS coronavirus (SARS-CoV-1), MERS coronavirus, COVID-19 coronavirus (SARS-CoV-2)); Orthomyxoviridae viruses (e.g., influenza virus (influenza virus type A, influenza virus type B, influenza virus type C, etc.)); Rhabdoviridae viruses (e.g., rabies virus); Bunyaviridae viruses (e.g., Crimean-Congo fever virus); Paramyxoviridae viruses (e.g., measles virus, human respiratory syncytial virus); and Filoviridae viruses (e.g., Ebola virus). When an enveloped RNA virus is to be tested, from the viewpoint that the present invention is more effective, the virus is preferably a Coronaviridae virus or an Orthomyxoviridae virus, and more preferably SARS-CoV-1, SARS-CoV-2, influenza virus type A, or influenza virus type B.

Examples of non-enveloped RNA viruses include Astroviridae viruses (e.g., Astrovirus); Caliciviridae viruses (e.g., Sapovirus, Norovirus); Picornaviridae viruses (e.g., Hepatitis A virus, Echovirus, Enterovirus, Coxsackievirus, Poliovirus, Rhinovirus); Hepeviridae viruses (e.g., Hepatitis E virus); and Reoviridae viruses (e.g., Rotavirus). When a non-enveloped RNA virus is to be tested, from the viewpoint that the present invention is more effective, Caliciviridae viruses and Reoviridae viruses are preferable, and Norovirus, Sapovirus, and Rotavirus are more preferable.

The present invention may be practiced to amplify nucleic acid from any of the above RNA viruses, but in one embodiment, from the viewpoint of more reliably obtaining the effects of the present invention, it may be practiced to detect preferably the RNA of a non-enveloped virus, more preferably the RNA of a Caliciviridae virus, and even more preferably the RNA of a norovirus. As shown in the results of the test examples described below, according to the present invention, even in one-step RT-PCR from low-copy norovirus RNA, non-specific reactions can be effectively suppressed, making it possible to detect the norovirus G1 gene and G2 gene with high sensitivity.

In the present invention, the concentration of template RNA added to the one-step RT-PCR reaction solution is, for example, 1 to 10,000 pg/μL, preferably 10 to 1,000 pg/μL, and more preferably 1 to 100 pg/μL, but is not limited to these.

In a particular preferred embodiment, in the one-step RT-PCR reaction of the present invention, a thermostable DNA polymerase having reverse transcription activity belonging to family A is used as the DNA polymerase. Here, the DNA polymerase having reverse transcription activity is a DNA polymerase that has both the ability to convert RNA to cDNA and the ability to amplify DNA, and by using such a DNA polymerase, it may be possible to perform a one-step RT-PCR reaction of a single enzyme system. Here, heat resistance means that the enzyme activity is not reduced by more than half even when heat treatment is performed at 70°C for 1 minute or more. As DNA polymerases used in PCR, DNA polymerases belonging to family A derived from thermophilic bacteria (also called pol I type) and DNA polymerases belonging to family B derived from hyperthermophilic archaea (also called α type) are conventionally known.

Specifically, examples of thermostable polymerases having reverse transcription activity belonging to family A that can be used in the present invention include, but are not limited to, DNA polymerase derived from Thermus thermophilus HB8 (Tth polymerase), DNA polymerase derived from Thermus sp. Z05 (Hawk Z05 polymerase), DNA polymerase derived from Thermotoga maritima (Tma polymerase), DNA polymerase derived from Bacillus caldotenax (Bca polymerase), and DNA polymerase derived from Bacillus stearothermophilus (Bst polymerase), and may be mutants in which the reverse transcription activity and thermostable DNA polymerase activity are not lost. Preferably, a DNA polymerase having reverse transcription activity selected from the group consisting of Tth, Z05, and mutants thereof is used. Among them, at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof is preferred, and by using these, even when a sample containing a large amount of insoluble substances is used, it is possible to detect RNA viruses with higher sensitivity. The amino acid sequence of Tth polymerase (SEQ ID NO: 1) and the amino acid sequence of Hawk Z05 polymerase (SEQ ID NO: 2) that can be particularly preferably used in the present invention are shown in the sequence table. In the present invention, mutant DNA polymerases (DNA polymerase mutants) in which some amino acids are modified based on these amino acid sequences without losing the effect can also be preferably used.

In this specification, a mutant of a thermostable DNA polymerase having reverse transcription activity refers to a mutant that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 99% or more sequence identity with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived, and has the activity of converting RNA to cDNA and the activity of amplifying DNA in the same manner as the wild-type DNA polymerase. Here, the method for calculating the identity of the amino acid sequence can be performed by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). As an example, it is possible to calculate the identity of the amino acid sequence by using the default (initial setting) parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/BLAST/. Furthermore, the mutant that can be used in the present invention may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and/or added (hereinafter, these are also collectively referred to as "mutation") in the amino acid sequence of the wild-type DNA polymerase from which it is derived, and may have the activity of converting RNA to cDNA and the activity of amplifying DNA, similar to the wild-type DNA polymerase. Here, "one or several" may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, and even more preferably 1 to 5, but is not particularly limited thereto.

In a specific embodiment, the total amount of the heat-stable DNA polymerase (preferably a heat-stable DNA polymerase having reverse transcriptase activity belonging to family A) contained in the one-step RT-PCR reaction solution is not particularly limited as long as the effect of the present invention is exhibited, but as an example, it is sufficient to be at least 2.0 ng/μL or more, preferably 3.0 ng/μL or more, and more preferably 4.2 ng/μL or more. The upper limit of the total amount of the heat-stable DNA polymerase contained in the one-step RT-PCR reaction solution is not particularly limited, but as an example, it can be 20 ng/μL or less, and may be 10 ng/μL or less, and the effect of the present invention can be sufficiently obtained even if it is, for example, 6 ng/μL or less. Therefore, it is preferable that the composition for use in the one-step RT-PCR reaction of the present invention contains the heat-stable DNA polymerase (preferably a heat-stable DNA polymerase having reverse transcriptase activity belonging to family A) in an amount adjusted so that the final concentration in the one-step RT-PCR reaction solution is the above concentration. The amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be roughly calculated from the safety data sheet (SDS). When a protein such as BSA is contained, it is preferable to calculate by the latter method.

In one embodiment of the present invention, a method for amplifying a template RNA in a sample while suppressing non-specific nucleic acid amplification is characterized by comprising at least the following steps:
(1) mixing a sample containing a template RNA with a one-step RT-PCR reaction solution containing the nucleic acid polymer to prepare a mixture;
(2) After sealing the reaction vessel, a one-step RT-PCR reaction is carried out.
Here, the step (1) preferably contains a heat-stable DNA polymerase having reverse transcription activity, which may enable a one-step RT-PCR reaction to be carried out in a single enzyme system.
The steps (1) and (2) are preferably carried out in the same vessel. That is, it is preferable not to transfer all or a part of the mixed liquid to another vessel between steps (1) and (2). Furthermore, in step (2), it is preferable not to open or close the lid of the reaction vessel after sealing the reaction vessel.

The RT-PCR cycle in step (2) consists of two steps: 1. reverse transcription reaction, and 2. PCR. Before and after each step, a heat treatment step for activating the hot start enzyme may be included. The temperature of the reverse transcription reaction in step 1 is determined by the reverse transcription activity of the heat-resistant DNA polymerase and the Tm value of the primer and the probe, and may be at least 25°C or higher. More preferably, it is 37°C or higher. In step 2, PCR may include three steps: [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of the primer to the template single-stranded DNA, and [3] extension of the primer using DNA polymerase. [2] and [3] may be performed at the same temperature to form a two-step process. In order to perform rapid RT-PCR, it is desirable to set the measurement program of the thermal cycler used in the RT-PCR reaction so that the extension times of the steps [2] and [3] combined are 60 seconds or less, preferably 45 seconds or less, and more preferably 30 seconds or less. In this specification, the term "PCR extension time" refers to the temperature set in a thermal cycler.

In order to enhance the effect of suppressing non-specific reactions, the one-step RT-PCR reaction solution used in the present invention is preferably used in combination with an anti-DNA polymerase antibody or by introducing a heat-labile blocking group into the DNA polymerase by chemical modification, thereby suppressing the enzymatic activity of the DNA polymerase before performing the one-step RT-PCR reaction, and thus enabling application to hot-start PCR.

The one-step RT-PCR reaction solution used in the present invention may contain, in addition to the heat-stable DNA polymerase, a nucleic acid polymer, a buffer, an appropriate salt, a magnesium salt or a manganese salt, deoxynucleotide triphosphates, a primer pair corresponding to the detection target region of the template RNA to be detected, and may further contain additives as necessary. Therefore, the composition for use in the one-step RT-PCR reaction of the present invention may also contain any of the above-mentioned components.

The buffer used in the present invention is not particularly limited, but examples thereof include Tris, Tricine, Bis-Tricine, and Bicine. The pH is adjusted to 6 to 9, more preferably 7 to 9, using sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, etc. The concentration of the buffer added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added to create ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and ammonium acetate.

The dNTPs used as substrates in the present invention are dATP, dCTP, dGTP, and dTTP, each added at 0.1 to 0.5 mM, most commonly about 0.2 mM. Preventive measures against cross-contamination may be taken by using dUTP instead of and/or as part of dTTP. When preventive measures against cross-contamination are taken, it is preferable to include Uracil-N-glycosylase (UNG).

Furthermore, in the present invention, it is preferable that the one-enzyme one-step RT-PCR reaction solution contains a divalent cation. By containing a divalent cation in this way, more stable and highly sensitive detection is possible. The divalent cation is not particularly limited, but examples thereof include magnesium ions, manganese ions, calcium ions, copper ions, iron ions, nickel ions, and zinc ions. It is preferable that the divalent cation contains magnesium ions and manganese ions. In the present invention, when magnesium ions or manganese ions are added to the one-enzyme one-step RT-PCR reaction solution, magnesium or manganese may be added, or a salt thereof may be added. Examples of magnesium or a salt thereof include magnesium, magnesium chloride, magnesium sulfate, and magnesium acetate, and examples of manganese or a salt thereof include manganese, manganese chloride, manganese sulfate, and manganese acetate. It is preferable that such magnesium, manganese, or a salt thereof is added to the RT-PCR reaction solution at about 1 to 10 mM. In the method of the present invention for detecting template RNA with suppressed nonspecific nucleic acid amplification, it is preferable to contain manganese or a salt thereof, from the viewpoint of easily obtaining a stable high sensitivity. In a specific embodiment, the one-step RT-PCR reaction solution preferably contains 1 mM or more of manganese or a salt thereof, preferably contains 1.5 mM or more of manganese or a salt thereof, and more preferably contains 2.0 mM or more of manganese or a salt thereof.

Additives contained in the one-step RT-PCR reaction solution may further include at least one selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), albumin (e.g., bovine serum albumin, etc.), sericin, BPF, glycerol, glycol, gelatin (e.g., fish gelatin, porcine gelatin, etc.), and surfactants. Of these, it is preferable to contain albumin and/or gelatin, and it is particularly preferable to contain both albumin and gelatin, and in particular it is preferable to contain bovine serum albumin and fish gelatin and/or porcine gelatin.

When the one-step RT-PCR reaction solution contains bovine serum albumin (BSA), its concentration is not particularly limited, but is preferably at least 0.5 mg/ml or more, more preferably at least 1 mg/ml or more, relative to the entire RT-PCR reaction solution. In samples with many impurities, a bovine serum albumin concentration of preferably 2 mg/ml or more, more preferably 3 mg/ml or more, enables good detection. The upper limit is not particularly limited, but as an example, it can be 10 mg/ml or less.

When the one-step RT-PCR reaction solution contains gelatin (e.g., fish gelatin, pork gelatin), its concentration is not particularly limited, but is preferably at least 0.5 mg/ml or more, more preferably at least 1 mg/ml or more, and even more preferably 5 mg/ml or more, relative to the entire RT-PCR reaction solution. In samples with many impurities, a gelatin concentration of preferably 7.5 mg/ml or more, and even more preferably 15 mg/ml or more, enables good detection. The upper limit is not particularly limited, but as an example, it can be 30 mg/ml or less.

Examples of surfactants contained in the one-step RT-PCR reaction solution include, but are not limited to, Triton X-100, Triton X-114, Tween 20, Nonidet P40, Briji 35, Briji 58, SDS, CHAPS, CHAPSO, and Emulgen 420. The concentration of the surfactant in the RT-PCR reaction solution is also not particularly limited, but is preferably 0.0001% or more, more preferably 0.002% or more, and even more preferably 0.005% or more to enable good detection. The upper limit is not particularly limited, but as an example, it can be 0.1% or less.

Examples of the betaine-like quaternary ammonium contained in the one-step RT-PCR reaction solution include, but are not limited to, betaine (trimethylglycine) and carnitine. The betaine structure is a compound that has stable positive and negative charges within the molecule, and is known to exhibit surfactant-like properties and promote nucleic acid amplification by DNA polymerase. The preferred concentration of the betaine-like quaternary ammonium is 0.1M to 2M, more preferably 0.2M to 1.2M.

Furthermore, it may be used in combination with substances known in the art to promote RT-PCR. Examples of promoters useful in the present invention include, but are not limited to, glycerol, polyols, protease inhibitors, single strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetate-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), and polyethylene glycol. In order to further reduce reaction inhibition, a chelating agent such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) may be included.

The primer pair used in the present invention includes two pairs of primers, one of which is complementary to the DNA extension product of the other primer. Another embodiment includes so-called multiplex PCR, in which two or more pairs of the above primers are included. Furthermore, when the target nucleic acid is a subtype, a degenerate primer may be included. The concentration of the primer to be detected is not particularly limited, but it is preferable that the concentration of the forward primer is 0.1 μM to 3 μM and the concentration of the reverse primer is 0.1 μM to 3 μM with respect to the entire RT-PCR reaction solution. More preferably, the concentration of the forward primer is 0.1 μM to 2 μM and the concentration of the reverse primer is 0.5 μM to 2 μM.

In another aspect, the present invention is a detection method further comprising at least one type of labeled hybridization probe or double-stranded DNA-binding fluorescent compound. This allows the analysis of the amplification product to be monitored by monitoring a fluorescent signal rather than by normal electrophoresis, reducing the analysis effort. Furthermore, there is no need to open the reaction vessel, which reduces the risk of contamination. When targeting multiple types of template RNA, it is also possible to separately identify the amplification of multiple types of nucleic acids by labeling each hybridization probe with a different fluorescent dye.

Examples of double-stranded DNA binding fluorescent compounds include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (registered trademark; Biotium), LCGreen (Idaho), and LightCycler (registered trademark) 480 ResoLight (Roche Applied Science).

Examples of hybridization probes used in the present invention include TaqMan hydrolysis probes (U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,538,848, U.S. Pat. No. 5,487,972, U.S. Pat. No. 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801), and FRET hybridization probes (WO 97/46707, WO 97/46712, WO 97/46714). When the target nucleic acid is a subtype, it may contain a degenerate sequence. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less. More preferably, it is 0.013 μM or more and 0.75 μM or less, and even more preferably, it is 0.02 μM or more and 0.5 μM or less.

In the present invention, the method of determining whether or not the amplification of nonspecific nucleic acids is suppressed can be confirmed by, but not limited to, subjecting the amplified product after RT-PCR to electrophoresis and comparing it with the band estimated from the number of bases of the template RNA. In electrophoresis, the amount of nonspecific nucleic acid amplification can be qualitatively determined based on the strength of the electrophoretic band. Another embodiment is a detection method including a double-stranded DNA binding fluorescent compound. By performing melting curve analysis in this method, it is possible to distinguish between the amplification of the target nucleic acid and the amplification of nonspecific nucleic acid from the difference in the Tm value of the amplified product. By comparing the height of the peak in the melting curve, it is possible to determine the amount of nonspecific nucleic acid amplification. In yet another embodiment, it can also be confirmed by a detection method using a hybridization probe (for example, a TaqMan hydrolysis probe). In a TaqMan hydrolysis probe detection system, the fluorescence intensity increases according to the amount of nucleic acid amplification, and the number of PCR reaction cycles at which the threshold fluorescence intensity is reached is called the Ct value. An increase in nonspecific nucleic acid amplification causes a decrease in the reached fluorescence intensity. Therefore, the amount of non-specific nucleic acid amplification can be determined by comparing the achieved fluorescence intensities.

Another aspect of the present invention is a composition for amplifying nucleic acid by one-step RT-PCR while suppressing non-specific nucleic acid amplification from template RNA in a sample. In a specific preferred aspect, the composition is for use in one-step RT-PCR of a single enzyme system, characterized by containing a specific nucleic acid polymer and a thermostable DNA polymerase having reverse transcription activity belonging to family A. The present invention may also be provided in the form of a kit for use in one-step RT-PCR in which non-specific nucleic acid amplification is suppressed, comprising these compositions.

The type and amount of the specific nucleic acid polymer, the type and amount of the thermostable DNA polymerase having reverse transcription activity belonging to Family A that may be optionally blended, the type and amount of the primer or probe, and the template RNA to be amplified used in these embodiments may be the same as those described in detail in the above-mentioned RNA testing method. These compositions or kits of the present invention may be accompanied by, for example, instructions for use that explain the method of use of the present invention. For example, the kit of the present invention may be provided in a form in which a specific nucleic acid polymer, a thermostable DNA polymerase having reverse transcription activity belonging to Family A, and other components are enclosed in the same container or in separate containers, and packaged in, for example, a single package, and includes information on the method of use of the kit.

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

Test Example 1. Study of one-enzyme one-step RT-PCR when potassium polyinosinate (inosinic acid polymer) is added 1
(1) Reaction solution The reaction solution had the following basic composition, and norovirus RNA was detected in the reaction solution by one-enzyme one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A and has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer solution (dilute 10x primer solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
Probe solution (dilute 10x probe solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
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 the above reagents, potassium polyinosinate* was added to a final concentration of 0 ng/μL to 100 ng/μL, and the RT-PCR reaction solution was adjusted to a final volume of 49 μL. 1 μL of norovirus RNA adjusted to 1250 to 25 copies/μL was added, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
* The base length distribution of the potassium polyinosinate used in this example was analyzed using a DNA-12000 reagent kit (manufactured by Shimadzu Corporation) with a microchip electrophoresis device (manufactured by the same company). As shown in FIG. 1, the potassium polyinosinate was found to have a peak base length of approximately 450 bases and to contain polyinosinic acid having a base length of approximately 150 to 8000 bases (reference value).
(2) Results The results are shown in Figures 6 to 7. Detection of up to 25 copies/reaction of both G1 and G2 noroviruses was confirmed under each condition. As the concentration of potassium polyinosinate added increased, the achieved fluorescence intensity increased, suggesting that nonspecific amplification was reduced.

Test Example 2. Study of one-enzyme one-step RT-PCR when potassium polyinosinate (inosinic acid polymer) is added 2
(1) Reaction solution The reaction solution had the following basic composition, and norovirus RNA was detected in the reaction solution by one-enzyme one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A and has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer solution (dilute 10x primer solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
Probe solution (dilute 10x probe solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
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 the above-mentioned reagents, the same potassium polyinosinate as used in Example 1 was added to a final concentration of 0 ng/μL to 500 ng/μL, and an RT-PCR reaction solution was prepared so that the final solution volume was 49 μL. Norovirus RNA adjusted to 1250 to 25 copies/μL was added in 1 μL increments, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
(2) Results The results are shown in Figures 8 to 9. Detection of up to 25 copies/reaction of both G1 and G2 noroviruses was confirmed under each condition. As the concentration of potassium polyinosinate added increased, the achieved fluorescence intensity was confirmed to increase, suggesting that nonspecific amplification was reduced even at a concentration of 500 ng/μL.

Test Example 3. Study of one-enzyme one-step RT-PCR when potassium polycytidylate (cytidylic acid polymer) and potassium polyadenylate (adenylic acid polymer) were added (1) Reaction solution The reaction solution shown below was used as the basic composition, and norovirus RNA was detected in the reaction solution in one-enzyme one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A and has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer solution (dilute 10x primer solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
Probe solution (dilute 10x probe solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
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 the above reagents, potassium polycytidylate* (Sigma, enzymatic synthesis product) was added to give final concentrations of 0 ng/μL, 200 ng/μL, and 300 ng/μL, and potassium polyadenylate* (Sigma, enzymatic synthesis product) was added to give final concentrations of 0 ng/μL and 100 ng/μL, and an RT-PCR reaction solution was prepared so that the final solution volume was 49 μL. 1 μL of norovirus RNA adjusted to a copy number of 1250 copies/μL was added to each solution, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
* The base length distribution of the potassium polycytidylic acid used in this example was analyzed using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation) with a microchip electrophoresis device (manufactured by the same company). As shown in FIG. 2, potassium polycytidylic acid had peak base lengths of approximately 200 bases and approximately 350 bases, and was found to contain polycytidylic acid of approximately 100 to 500 bases in length (reference values).
* The base length distribution of the potassium polyadenylate used in this example was analyzed using a microchip electrophoresis device (manufactured by Shimadzu Corporation) and a DNA-12000 reagent kit. As shown in FIG. 3, potassium polyadenylate was found to have a peak base length of approximately 300 bases and to contain polyadenylic acid of approximately 100 to 5,000 bases (reference value).
(2) Results The results when potassium polyadenylate was added are shown in Figures 10 and 11. The addition of potassium polyadenylate did not have a significant inhibitory effect on G1 gene detection, but an increase in the achieved relative fluorescence intensity was confirmed in G2 gene detection upon addition, confirming that nonspecific nucleic acid amplification was suppressed. The results when potassium polycytidylate was added are shown in Figures 12 and 13. With potassium polycytidylate, an increase in the achieved relative fluorescence intensity was confirmed at 200 to 300 ng/μL for both G1 and G2 genes, confirming that nonspecific nucleic acid amplification was suppressed.

Test Example 4. Study of one-enzyme one-step RT-PCR when polydeoxythymidylic acid (thymidylic acid polymer) and potassium polyguanylate (guanylic acid polymer) were added (1) Reaction solution The reaction solution shown below was used as the basic composition, and norovirus RNA in the reaction solution was detected in one-enzyme one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A and has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer solution (dilute 10x primer solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
Probe solution (dilute 10x probe solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
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 the above reagents, potassium polyguanylate* (Sigma, enzymatic synthesis product) and polydeoxythymidylic acid (approximately 70 base length) (Hokkaido System Science, chemical synthesis product) were added to a final concentration of 0 ng/μL to 10 ng/μL to prepare an RT-PCR reaction solution with a final volume of 49 μL. Norovirus RNA with a copy number of 1250 copies/μL was added in 1 μL increments, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
* The base length distribution of the potassium polyguanylate used in this example was analyzed using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation) with a microchip electrophoresis device (manufactured by the same company). As shown in FIG. 4, it was found that the potassium polyguanylate had a peak base length of approximately 100 bases and contained polyguanylic acid having a length of approximately 100 to 600 bases (reference value).
(2) Results The results are shown in Figures 14 and 15. When polydeoxythymidylic acid and potassium polyguanylate were added, an increase in the achieved relative fluorescence intensity was confirmed for both G1 and G2 genes, confirming that nonspecific nucleic acid amplification was suppressed.

Test Example 5. Study of one-enzyme one-step RT-PCR when polyinosinic acid-sodium polycytidylate (double-stranded inosinic acid cytidylic acid polymer) was added (1) Reaction solution The reaction solution shown below was used as the basic composition, and norovirus RNA in the reaction solution was detected in one-enzyme one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A and has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer solution (dilute 10x primer solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
Probe solution (dilute 10x probe solution to 1x)
(Norovirus Detection Kit G1/G2 - High-Speed Probe Detection - (Toyobo) Attachment)
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 the above-mentioned reagents, polyinosinic acid-polycytidylic acid sodium salt (Poly(I:C)) was added to give final concentrations of 0 ng/μL and 100 ng/μL, and an RT-PCR reaction solution was prepared so that the final solution volume was 49 μL. 1 μL of norovirus RNA, the copy number of which had been adjusted to 1250 copies/μL, was added to each solution, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
* The base length distribution of the polyinosinic acid-sodium polycytidylate (Poly(I:C)) used in this example was analyzed using a reagent kit DNA-12000 (manufactured by Shimadzu Corporation) with a microchip electrophoresis device (manufactured by the same company). As shown in FIG. 5, it was found that polyinosinic acid-sodium polycytidylate (Poly(I:C)) had peak base lengths of approximately 100 bases and approximately 150 bases, and contained polyinosinic acid and polycytidylic acid with lengths of approximately 50 to 800 bases (reference values).
(2) Results The results are shown in Figures 16 to 17. For both norovirus G1 and G2, an improvement in the achievable relative fluorescence intensity was confirmed at a Poly(I:C) concentration of 200 ng/μL, and it was confirmed that nonspecific nucleic acid amplification was reduced.

Test Example 6. Study of one-enzyme one-step RT-PCR when potassium polyinosinate (inosinic acid polymer) is added (SARS-CoV-2 RNA detection)
(1) Reaction solution The reaction solution shown below was used as the basic composition, and SARS-CoV-2 RNA (regions 1 and 2 of the N gene) in the reaction solution was detected in a one-enzyme, one-step RT-PCR. The rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to family A that has reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer/probe solution (dilute 10x primer solution to 1x)
(Included with the New Coronavirus Detection Kit -Multi- (Toyobo))
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 the above-mentioned reagents, the same potassium polyinosinate as used in Example 1 was added to a final concentration of 0 ng/μL to 200 ng/μL, and an RT-PCR reaction solution was prepared so that the final solution volume was 49 μL. 1 μL of SARS-CoV-2 RNA (manufactured by Twist Bioscience) adjusted to 1250 to 25 copies/μL was added, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
(2) Results The results are shown in Figures 18 and 19. Under each condition, detection of up to 25 copies/reaction was confirmed for both region 1 (Cy5) and region 2 (ROX) in the N gene of SARS-CoV-2. As the concentration of potassium polyinosinate added increased, the achieved fluorescence intensity was confirmed to increase, suggesting that non-specific amplification was reduced.

Test Example 7. Examination of one-enzyme one-step RT-PCR when potassium polyinosinate (inosinic acid polymer) is added (detection of influenza virus RNA)
(1) Reaction solution The reaction solution had the following basic composition, and influenza virus type A or type B RNA in the reaction solution was detected by one-enzyme, one-step RT-PCR. The rTth DNA polymerase used in this test example is a heat-resistant DNA polymerase belonging to family A and having reverse transcription activity.
Reaction solution (dilute 10x Buffer to 1x)
(Included with rTaq DNA Polymerase 10x Buffer (Toyobo))
Primer-probe ^* (Use a primer-probe set for influenza A or B)
[Influenza A primer-probe set]
0.2 μM InfA For1
0.2 μM InfA For2
0.2 μM InfA Rev1
0.2 μM InfA Rev2
0.2 μM InfA Probe (ROX)
[Influenza B primer-probe set]
0.2 μM InfB For
0.2 μM InfB Rev
0.2 μM InfA Probe (HEX)
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 the above-mentioned reagents, the same potassium polyinosinate as used in Example 1 was added to a final concentration of 0 ng/μL to 200 ng/μL, and an RT-PCR reaction solution was prepared so that the final solution volume was 49 μL. 1 μL of influenza virus type A or type B RNA (manufactured by Twist Bioscience) adjusted to 1250 to 25 copies/μL was added, and RT-PCR was performed in a 50 μL reaction system.
This was used to carry out real-time PCR reaction using BioRad's CFX96WELL DEEP with the following temperature cycle: Fluorescence reading was performed at 52°C and 40 cycles of extension step, and the achieved relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
90°C 1 minute (heat treatment conditions)
58°C 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)
* The primer and probe sequences used in this example were primer and probe sets for detecting influenza types A and B described in "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes" published by the U.S. Centers for Disease Control and Prevention (CDC).
(2) Results The results are shown in Figures 20 and 21. Detection of up to 25 copies/reaction of influenza A and B genes was confirmed under each condition. As the concentration of potassium polyinosinate added increased, the achieved fluorescence intensity was confirmed to increase, suggesting that non-specific amplification was reduced.

The present invention is suitable for use in molecular biology research, as well as in tests for clinical testing and food hygiene management.

Claims (20)

A method for amplifying nucleic acid from template RNA by one-step RT-PCR, comprising suppressing non-specific nucleic acid amplification by using at least one type of nucleic acid polymer selected from the group consisting of inosinic acid polymer, cytidylic acid polymer, guanylic acid polymer, adenylic acid polymer, thymidylic acid polymer, uridylic acid polymer, deoxyinosinic acid polymer, deoxycytidylic acid polymer, deoxyguanylic acid polymer, deoxyadenylic acid polymer, deoxythymidylic acid polymer, and deoxyuridylic acid polymer,
The one-step RT-PCR is a one-enzyme type one-step RT-PCR . The method according to claim 1, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polyadenylic acid, polythymidylic acid, polyuridylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, polydeoxyadenylic acid, polydeoxythymidylic acid, polydeoxyuridylic acid, and salts thereof. The method according to claim 1 or 2, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polycytidylic acid, polyguanylic acid, polydeoxyinosinic acid, polydeoxycytidylic acid, polydeoxyguanylic acid, and salts thereof. The method according to any one of claims 1 to 3, wherein the nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof. The method according to any one of claims 1 to 4, wherein the nucleic acid polymer comprises a nucleic acid polymer having a total length of 30 to 10,000 bases. The method according to any one of claims 1 to 5, wherein the nucleic acid polymer is allowed to coexist in a one-step RT-PCR reaction solution at a final concentration of 0.1 ng/μL or more and 1000 ng/μL or less. The method according to any one of claims 1 to 6, wherein the DNA polymerase used in the one-step RT-PCR is a thermostable DNA polymerase having reverse transcription activity belonging to family A. The method according to claim 7, characterized in that the thermostable DNA polymerase having reverse transcription activity belonging to family A is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof. The method according to claim 8, wherein the mutant has an amino acid sequence that is 90% or more identical to the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2) and exhibits reverse transcription activity and thermostable DNA polymerase activity. The method according to claim 8 or 9, wherein the mutant has an amino acid sequence having one or several amino acid deletions, substitutions and/or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2), and exhibits reverse transcription activity and thermostable DNA polymerase activity. The method according to any one of claims 1 to 10, wherein the template RNA is RNA derived from a biological sample, an environmental sample, or a cell. The method according to claim 11, wherein the biological sample is at least one selected from the group consisting of feces, urine, vomit, saliva, sputum, throat swab, nasal swab, nasal mucus, blood, plasma, and serum. The method according to claim 11, wherein the environmental sample is at least one selected from the group consisting of a swab test sample, a soil sample, and a sewage sample. A composition for use in a one-step, single-enzyme RT-PCR reaction in which non-specific nucleic acid amplification is suppressed, comprising at least one type of nucleic acid polymer selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers , deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers. The composition according to claim 14, wherein the nucleic acid polymer has a total length of 30 to 10,000 bases. The composition according to claim 14 or 15, wherein the nucleic acid polymer is prepared so that the final concentration in the one-step RT-PCR reaction solution is 0.1 ng/μL or more and 1000 ng/μL or less. The composition according to any one of claims 14 to 16, further comprising a thermostable DNA polymerase having reverse transcription activity belonging to family A. The composition according to claim 17, characterized in that the thermostable DNA polymerase having reverse transcription activity belonging to family A is at least one thermostable DNA polymerase having reverse transcription activity selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and mutants thereof. The composition according to any one of claims 14 to 18, which is used for amplifying nucleic acids from RNA derived from a biological sample, an environmental sample, or a cell. 19. A kit for use in a one-enzyme, one-step RT-PCR reaction in which non-specific nucleic acid amplification is suppressed, comprising the composition according to any one of claims 14 to 18.