WO2021177041A1 - Procédé pour supprimer l'amplification non spécifique des acides nucléiques - Google Patents

Procédé pour supprimer l'amplification non spécifique des acides nucléiques Download PDF

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WO2021177041A1
WO2021177041A1 PCT/JP2021/006071 JP2021006071W WO2021177041A1 WO 2021177041 A1 WO2021177041 A1 WO 2021177041A1 JP 2021006071 W JP2021006071 W JP 2021006071W WO 2021177041 A1 WO2021177041 A1 WO 2021177041A1
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acid
polymer
nucleic acid
acid polymer
mol
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謙太 寺内
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東洋紡株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to the generation and amplification of nucleic acid amplification.
  • the present invention uses compositions and methods useful for the production and amplification of nucleic acids from ribonucleic acid (RNA) templates, specifically the production and amplification of nucleic acids by reverse transcription reactions using specific nucleic acid polymers. Concerning the composition and method for carrying out. More specifically, it relates to the production and amplification of nucleic acids by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the presence of a specific nucleic acid polymer.
  • RT-PCR real-time reverse transcription-polymerase chain reaction
  • the method of the present invention makes it possible to specifically detect RNA derived from RNA virus in fecal samples, blood samples, environmental wiping samples, etc., and biological RNA in biological samples, for example.
  • the present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.
  • Nucleic acid amplification is a technology that amplifies several copies of a target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more. It is also widely used in the microbiological examination of.
  • a typical nucleic acid amplification method is PCR (Polymerase Chain Reaction).
  • PCR includes (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) the primer using a DNA polymerase. This is a method in which the target nucleic acid in the sample is amplified by repeating the three steps of extension as one cycle. Annealing and elongation may be performed in two steps at the same temperature.
  • RT reverse transcription
  • This RT-PCR is a one-step RT-PCR in which (1) RT and PCR are carried out discontinuously, and (2) RT and PCR are carried out in succession using a single enzyme.
  • -PCR (3) It is roughly divided into three types: two-enzyme system 1-step RT-PCR in which RT and PCR are continuously performed using two types of enzymes, reverse transcription enzyme and DNA polymerase.
  • RT-PCR in genetic testing and virus testing, one-step RT-PCR is preferred because of its high processing capacity and avoiding contamination due to opening and closing of the reaction vessel during the reaction.
  • Two-enzyme system In one-step RT-PCR, at least two types of enzymes, reverse transcriptase and DNA polymerase, are used.
  • a DNA polymerase having reverse transcription activity such as Tth DNA polymerase is used.
  • the detection sensitivity of 1-step RT-PCR is greatly related to the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase. So far, in order to improve the reverse transcription efficiency of reverse transcriptase and the DNA synthesis efficiency of DNA polymerase, the use of amino acid variants with improved enzyme activity (Patent Document 1), various additives, etc. has been studied (Patent Document 1). Patent Document 2). In the two-enzyme system 1-step RT-PCR, it is also known that the reverse transcriptase itself inhibits PCR, and a method of adding a nucleic acid polymer as a method of reducing the inhibition by the reverse transcriptase is also known (Patented). Document 3). On the other hand, in the one-enzyme system one-step RT-PCR, PCR is not inhibited by reverse transcriptase.
  • Non-specific gene amplification refers to the amplification of nucleic acid sequences other than the target gene in PCR.
  • dNTP deoxynucleotide triphosphate
  • the present invention has been made in view of the above-mentioned prior art, and in the case of nucleic acid amplification from template RNA by 1-step RT-PCR, it is possible to effectively suppress non-specific nucleic acid amplification while being a simple method. Is to provide a useful means of becoming.
  • the present inventors have conducted diligent research, and as a result, by using a specific nucleic acid polymer, non-specific nucleic acid amplification can be suppressed in 1-step RT-PCR, and the target nucleic acid can be highly sensitive. We have found that specific detection is possible, and have completed the present invention.
  • Typical inventions of the present application are as follows.
  • an inosinate polymer In a method of amplifying nucleic acid from a template RNA by one-step RT-PCR, an inosinate polymer, a thymidylate polymer, a guanylate polymer, an adenylate polymer, a thymidylate polymer, a uridylic acid polymer, a deoxyinosic acid polymer, and a deoxycyti.
  • nucleic acid polymer selected from the group consisting of dialic acid polymers, deoxyguanylate polymers, deoxyadenylate polymers, deoxythymidylate polymers, and deoxyuridilic acid polymers. how to.
  • the nucleic acid polymer is polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polytimidic acid, polyuridylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenic acid, polydeoxy.
  • the method according to Item 1 which is at least one homopolymer selected from the group consisting of thymidylic acid, polydeoxyuridylic acid, and salts thereof.
  • Item 2 The method according to Item 1 or 2, which is a polymer.
  • nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof.
  • nucleic acid polymer contains the nucleic acid polymer having a total length of 30 to 10000 bases.
  • nucleic acid polymer is allowed to coexist in an amount such that the final concentration is 0.1 ng / ⁇ L or more and 1000 ng / ⁇ L or less in the 1-step RT-PCR reaction solution.
  • 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 belonging to Family A and having reverse transcription activity.
  • the heat-resistant DNA polymerase belonging to the family A having reverse transcription activity is selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and variants thereof, and is at least one type of heat-resistant DNA having reverse transcription activity.
  • Item 6. The method according to Item 1 to 8, which is a polymerase.
  • the variant comprises an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2), and has reverse transcription activity and heat resistance.
  • Item 9. The method according to Item 9, which exhibits DNA polymerase activity.
  • the mutant comprises 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).
  • Item 9 or 10 which exhibits reverse transcription activity and heat-resistant DNA polymerase activity.
  • RNA RNA derived from a biological sample, an environmental sample, or a cell.
  • the biological sample is at least one selected from the group consisting of feces, urine, vomitus, saliva, sputum, pharyngeal swab, nasal swab, runny nose, blood, plasma, and serum.
  • the method according to 12. Item 14. The method according to Item 12, wherein the environmental sample is at least one selected from the group consisting of a wiping test sample, a soil sample, and a sewage sample.
  • the nucleic acid polymer is polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polytimidic acid, polyuridylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid, polydeoxy.
  • Item 2 The composition according to Item 15, which is at least one homopolymer selected from the group consisting of thymidylic acid, polydeoxyuridylic acid, and salts thereof.
  • nucleic acid polymer is at least one homopolymer selected from the group consisting of polyinosinic acid, polydeoxyinosinic acid, and salts thereof.
  • the nucleic acid polymer is selected from the group consisting of inosinic acid, cytidinelic acid, guanylic acid, deoxyinosinic acid, deoxycitidilic acid, deoxyguanylic acid, derivatives thereof, and salts thereof in all the constituent units.
  • Item 3. The method according to Item 1, or the composition according to Item 15, which comprises 60 mol% or more of a constituent unit derived from a nucleotide.
  • the nucleic acid polymer is selected from the group consisting of inosinic acid, cytidinelic acid, guanylic acid, deoxyinosinic acid, deoxycitidilic acid, deoxyguanylic acid, derivatives thereof, and salts thereof in all the constituent units.
  • nucleic acid polymer contains 60 mol% or more of the constituent units derived from nucleotides selected from the group consisting of inosinic acid, deoxyinocinic acid, derivatives thereof, and salts thereof in all the constituent units. , 19 or 20, or the composition according to item 15, 19 or 20.
  • nucleic acid polymer contains 90 mol% or more of the constituent units derived from nucleotides selected from the group consisting of inosinic acid, deoxyinosinic acid, derivatives thereof, and salts thereof in all the constituent units. , The method according to any one of 19 to 21, or the composition according to any one of Items 15, 19 to 21. Item 23.
  • composition according to any one of Items 15 to 22, wherein the nucleic acid polymer comprises the nucleic acid polymer having a total length of 30 to 10000 bases.
  • 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 the 1-step RT-PCR reaction solution is 0.1 ng / ⁇ L or more and 1000 ng / ⁇ L or less. .. Item 26.
  • the heat-resistant DNA polymerase belonging to the family A having reverse transcription activity is selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and variants thereof, and is at least one type of heat-resistant DNA having reverse transcription activity.
  • Item 28. The composition according to Item 26, which is a polymerase.
  • the variant comprises an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 1) or Hawk Z05 polymerase (SEQ ID NO: 2), and has reverse transcription activity and heat resistance.
  • the variant comprises 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).
  • the biological sample is at least one selected from the group consisting of feces, urine, vomitus, saliva, sputum, pharyngeal swab, nasal swab, runny nose, blood, plasma, and serum.
  • the present invention by coexisting a predetermined nucleic acid polymer in a reaction solution, non-specific nucleic acid amplification in a one-step RT-PCR reaction can be suppressed, thereby producing a specific amplification product of a target nucleic acid intended for amplification. Since the amount of production can be increased, it may be possible to improve the detection sensitivity.
  • One embodiment of the present invention is a method of amplifying nucleic acid by a 1-step RT-PCR reaction using RNA contained in a sample as a template, by performing a 1-step RT-PCR reaction in the coexistence of a specific nucleic acid polymer.
  • This is a method of suppressing non-specific nucleic acid amplification.
  • non-specific nucleic acid amplification means that a nucleic acid sequence other than the target sequence of the template RNA is synthesized by one-step RT-PCR.
  • a reverse transcription reaction (also referred to as RT reaction) for converting the template RNA into cDNA is first performed, and the cDNA obtained thereby is amplified by a PCR reaction.
  • the one-step RT-PCR refers to a method of amplifying nucleic acid from a template RNA by carrying out the above-mentioned RT reaction and PCR reaction continuously or in parallel.
  • the one-step RT-PCR includes a two-enzyme one-step RT-PCR reaction in which the RT reaction and the PCR reaction are continuously carried out using two separate types of enzymes (reverse transcriptase and DNA polymerase).
  • RT-PCR and the like of a one-enzyme system in which RT reaction and PCR reaction are carried out continuously or in parallel using a single enzyme DNA polymerase having both reverse transcriptase activity such as Tth DNA polymerase
  • DNA polymerase having both reverse transcriptase activity such as Tth DNA polymerase
  • the present invention can be carried out in any one-step RT-PCR, it is a one-enzyme system from the viewpoint of more reliably suppressing non-specific nucleic acid amplification and facilitating high-sensitivity detection of target nucleic acids. It is preferable to carry out by 1-step RT-PCR.
  • 1-step RT-PCR preferably carries out all reactions in the same reaction vessel.
  • a substrate such as deoxynucleotide triphosphate (dNTP) required for target nucleic acid amplification that should occur originally, etc. Is depleted, and the target nucleic acid cannot be amplified in a sufficient amount, which may lead to a decrease in detection sensitivity. Therefore, the method of the present invention capable of suppressing non-specific nucleic acid amplification in a one-step RT-PCR reaction can suppress a decrease in detection sensitivity of such a target nucleic acid. Therefore, the method of the present invention can also be, for example, a method of increasing the production amount of a specific nucleic acid amplification product, a method of improving the specific amplification of a target nucleic acid, or the like.
  • dNTP deoxynucleotide triphosphate
  • nucleic acid polymer used in the present invention examples include inosinate polymer, cytidilic acid polymer, guanylic acid polymer, adenylate polymer, thymidylate polymer, uridylic acid polymer, deoxyinosic acid polymer, deoxycitidilic acid polymer, deoxyguanylate polymer, and deoxy. At least one selected from the group consisting of adenylate polymer, deoxythymidylate polymer, and deoxyuridinelic acid polymer is preferable.
  • inosinic acid polymer from the viewpoint of suppressing non-specific nucleic acid amplification in 1-step RT-PCR even more effectively, inosinic acid polymer, citidilic acid polymer, guanylate polymer, deoxyinosinic acid polymer, deoxycitidilic acid polymer, and At least one selected from the group consisting of deoxyguanylate polymers is more preferred, and inosinic acid polymers and / or deoxyinosinic acid polymers are particularly preferred.
  • two or more kinds of nucleic acid polymers as described above may be used.
  • an inosinic acid polymer and a cytidine monophosphate polymer may be used in combination, or a deoxyinosinic acid polymer and a cytidine monophosphate polymer may be used in combination.
  • a deoxyinosinic acid polymer and a cytidine monophosphate polymer may be used in combination.
  • two or more kinds of nucleic acid polymers it is preferable that one of them is an inosinic acid polymer or a deoxyinosinic acid polymer.
  • each polymer will be described in detail.
  • Inosinic acid polymer is used to include inosinic acid homopolymers (polyinosinic acid), inosinic acid copolymers, derivatives thereof, and salts thereof. Further, a double-stranded nucleic acid polymer called poly (I: C) or the like in which an inosinic acid homopolymer (polyinosinic acid) and a cytidine monophosphate homopolymer (polysilicate) are annealed is known in the art.
  • the inosinic acid polymer also includes a double-stranded nucleic acid polymer in which at least one of the double strands is an inosinic acid polymer (for example, polyinosinic acid), such as the above-mentioned double-stranded nucleic acid polymer.
  • An inosinic acid copolymer is a polymer having a structural unit derived from inosinic acid (IMP) and a structural unit derived from a nucleotide other than IMP.
  • IMP inosinic acid
  • a nucleotide-derived structural unit such as IMP means a unit composed of nucleotides as represented by the following formula:
  • R is a hydrogen atom or hydroxyl
  • Base is a nucleobase such as inosine, cytosine, guanine, adenine, thymine, and uracil.
  • Nucleotides other than IMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides. Examples of nucleotides other than IMP include deoxyinocinic acid (dIMP), cytidine monophosphate (CMP), deoxycitidilic acid (dCMP), guanylic acid (GMP), deoxyguanylic acid (dGMP), adenylic acid (AMP), and deoxy.
  • dIMP deoxyinocinic acid
  • CMP cytidine monophosphate
  • dCMP deoxycitidilic acid
  • GMP guanylic acid
  • dGMP deoxyguanylic acid
  • AMP adenylic acid
  • Examples thereof include adenylic acid (dAMP), thymidylate (TMP), deoxythymidylate (dTMP), uridylic acid (UMP), deoxyuridilic acid (dUMP), and a combination of two or more of these.
  • dAMP adenylic acid
  • TMP thymidylate
  • dTMP deoxythymidylate
  • UMP uridylic acid
  • dUMP deoxyuridilic acid
  • dUMP deoxyuridilic acid
  • dUMP deoxyuridilic acid
  • the above-mentioned “derivative” refers to a derivative in which a part of the structure is modified while retaining the basic skeleton of the original polymer. Modifications include, for example, oxidation, reduction, atomic substitution, introduction of functional groups, and the like.
  • the above-mentioned “derivative” is a C1 having a functional group (for example, a fluorine atom, a bromine atom, an iodine atom, a lower alkyl group (for example, a methyl group, an ethyl group, etc.)) at a base portion in at least a part of the structural unit of the original polymer.
  • the constituent units derived from IMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units.
  • the constituent unit derived from IMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the IMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more.
  • the number is 70 or more, and for example, 100 or more or 150 or more may be used.
  • the number of repetitions of the IMP-derived structural unit may be, for example, 10,000 or less and 8,000 or less, and may be, for example, 5000 or less, 3000 or less, 1000 or less, and 800 or less.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Cytidine monophosphate polymer The cytidine monophosphate polymer is used in the sense of including a cytidine monophosphate homopolymer (polyphosphate), a cytidine monophosphate copolymer, derivatives thereof, and salts thereof. Further, a double-stranded nucleic acid polymer called poly (I: C) or the like in which an inosinic acid homopolymer (polyinosinic acid) and a cytidine monophosphate homopolymer (polysilicate) are annealed is known in the art.
  • the cytidinelic acid polymer also includes a double-stranded nucleic acid polymer in which at least one of the double strands is a cytidinelic acid polymer (for example, polycitidilic acid), such as the above-mentioned double-stranded nucleic acid polymer. ..
  • the cytidine monophosphate copolymer is a polymer having a constituent unit derived from CMP and a constituent unit derived from a nucleotide other than CMP. Nucleotides other than CMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • nucleotides other than CMP examples include IMP, dIMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these.
  • at least one selected from the group consisting of IMP, dIMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, dIMP, dCMP, GMP, and dGMP is at least one selected. More preferably, at least one selected from the group consisting of IMP and dIMP is more preferable.
  • the constituent units derived from CMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units. It is mol% or more, 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.
  • the constituent unit derived from CMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the CMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. Even more preferably, the number is 70 or more, and for example, 100 or more may be used.
  • the number of repetitions of the CMP-derived structural unit may be, for example, 10,000 or less, 8,000 or less, 5000 or less, 3000 or less, 1000 or less, 800 or less, or 500 or less.
  • the repetition may be continuous or intermittent, preferably continuous.
  • the guanylic acid polymer is used in the sense of including a guanylic acid homopolymer (polyguanylic acid), a guanylic acid copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a guanylic acid polymer (for example, polyguanylic acid) is also included in the guanylic acid polymer.
  • a guanylic acid copolymer is a polymer having a structural unit derived from GMP and a structural unit derived from a nucleotide other than GMP.
  • Nucleotides other than GMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than GMP include IMP, dIMP, CMP, dCMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, dGMP, AMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, and dGMP is at least one selected.
  • the constituent units derived from GMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units. It is mol% or more, 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.
  • the GMP-derived structural unit may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the structural units.
  • the number of repetitions of the GMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. Even more preferably, the number is 70 or more, and for example, 100 or more may be used.
  • the number of repetitions of the GMP-derived structural unit may be, for example, 10,000 or less, 8,000 or less, 5000 or less, 3000 or less, 1000 or less, or 600 or less.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Adenylate polymer is used in the sense of including adenylate homopolymer (polyadenylate), adenylate copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is an adenylate polymer (for example, polyadenylic acid) is also included in the adenylate polymer.
  • the adenylate copolymer is a polymer having a structural unit derived from AMP and a structural unit derived from a nucleotide other than AMP.
  • Nucleotides other than AMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than AMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • the constituent units derived from AMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units. It is mol% or more, 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.
  • the structural unit derived from AMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the structural units.
  • the number of repetitions of the AMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. Even more preferably, the number is 70 or more, and for example, 100 or more may be used.
  • the number of repetitions of the AMP-derived structural unit can be, for example, 10,000 or less, 8,000 or less, and 5,000 or less. The repetition may be continuous or intermittent, preferably continuous.
  • the thymidylate polymer is used to include a thymidylate homopolymer (polythymidylate), a thymidylate copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a thymidylate polymer (for example, polythymidylate) is also included in the thymidylate polymer.
  • the thymidylate copolymer is a polymer having a structural unit derived from TMP and a structural unit derived from a nucleotide other than TMP.
  • Nucleotides other than TMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than TMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, dTMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and it is selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • At least one selected from the group consisting of IMP and dIMP is more preferable.
  • the constituent units derived from TMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units. It is mol% or more, 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.
  • the constituent unit derived from TMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the TMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. , Even more preferably 70 or more.
  • the number of repetitions of the TMP-derived structural unit is, for example, 10000 or less, 8000 or less, 5000 or less, 3000 or less, 1000 or less, for example, less than 100, 95 or less, or 90 or less. could be.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Uridine monophosphate polymer The uridylic acid polymer is used in the sense of including a uridylic acid homopolymer (polyuridilic acid), a uridylic acid copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a uridylic acid polymer (for example, polyuridylic acid) is also included in the uridylic acid polymer.
  • 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.
  • Nucleotides other than UMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than UMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and it is selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • At least one selected from the group consisting of IMP and dIMP is more preferable.
  • the constituent units derived from UMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 75 in all the constituent units. It is mol% or more, 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.
  • the UMP-derived structural unit may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the structural units.
  • the number of repetitions of the UMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. More preferably, the number is 70 or more, and for example, 100 or more or 150 or more may be used.
  • the number of repetitions of the UMP-derived structural unit may be, for example, 10,000 or less, 8,000 or less, 5000 or less, 3000 or less, or 1000 or less. The repetition may be continuous or intermittent, preferably continuous.
  • Deoxyinosinic acid polymer is used in the sense of including deoxyinosinic acid homopolymer (polydeoxyinosinic acid), deoxyinosinic acid copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a deoxyinosinic acid polymer (for example, polydeoxyinosinic acid) is also included in the deoxyinosinic acid polymer.
  • the deoxyinosinic acid copolymer is a polymer having a constituent unit derived from dIMP and a constituent unit derived from a nucleotide other than dIMP.
  • Nucleotides other than dIMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than dIMP include IMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, CMP, dCMP, GMP, and dGMP is at least one selected. More preferably, IMP is more preferable.
  • the constituent units derived from dIMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 70 mol% or more, among all the constituent units. It is 75 mol% or more, 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.
  • the constituent unit derived from dIMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the structural unit derived from dIMP (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60.
  • even more preferably 70 or more for example, 100 or more, 150 or more may be used.
  • the number of repetitions of the constituent unit derived from dIMP may be, for example, 10,000 or less and 8,000 or less, and may be, for example, 5000 or less, 3000 or less, 1000 or less, and 800 or less.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Deoxycitidilic acid polymer The deoxycitidilic acid polymer is used in the sense of including a deoxycitidilic acid homopolymer (polydeoxycitidilic acid), a deoxycitidilic acid copolymer, derivatives thereof, and salts thereof.
  • the deoxycytidine diylate copolymer is a polymer having a constituent unit derived from dCMP and a constituent unit derived from a nucleotide other than dCMP.
  • Nucleotides other than dCMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides. Examples of nucleotides other than dCMP include IMP, dIMP, CMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these.
  • At least one selected from the group consisting of IMP, dIMP, CMP, GMP, dGMP, AMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, dIMP, CMP, GMP, and dGMP is at least one selected. More preferably, at least one selected from the group consisting of IMP and dIMP is more preferable.
  • Examples of the above-mentioned “derivative" and “salt” include those similar to those described in the above-mentioned "(1) Inosinic 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 70 mol% or more, among all the constituent units. Is 75 mol% or more, 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.
  • the structural unit derived from dCMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the structural units.
  • the number of repetitions of the structural unit derived from dCMP (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60.
  • the number is more than 70, more preferably 70 or more, and may be 100 or more, for example.
  • the number of repetitions of the structural unit derived from dCMP may be, for example, 10,000 or less, 8,000 or less, 5000 or less, 3000 or less, 1000 or less, 800 or less, or 500 or less.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Deoxyguanylic acid polymer is used to include deoxyguanylic acid homopolymers (polydeoxyguanylic acid), deoxyguanylic acid copolymers, derivatives thereof, and salts thereof.
  • the deoxyguanylate polymer also includes a double-stranded nucleic acid polymer in which at least one of the double strands is a deoxyguanylate polymer (for example, polydeoxyguanylic acid).
  • the deoxyguanyl acid copolymer is a polymer having a constituent unit derived from dGMP and a constituent unit derived from a nucleotide other than dGMP.
  • Nucleotides other than dGMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than dGMP include IMP, dIMP, CMP, dCMP, GMP, AMP, dAMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, AMP, and dAMP is preferable, and at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, and GMP is at least one selected.
  • the constituent units derived from dGMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 70 mol% or more, among all the constituent units.
  • the structural unit derived from dGMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the structural units.
  • the number of repetitions of the structural unit derived from dGMP (which can also be expressed as base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more.
  • the number is 70 or more, and for example, 100 or more may be used.
  • the number of repetitions of the structural unit derived from dGMP is, for example, 10000 or less, 8000 or less, preferably 6500 or less, more preferably 6000 or less, 5000 or less, 3000 or less, 1000 or less, 600. It can be less than one.
  • the repetition may be continuous or intermittent, preferably continuous.
  • Deoxyadenylic acid polymer The deoxyadenylic acid polymer is used in the sense of including a deoxyadenylic acid homopolymer (polydeoxyadenylic acid), a deoxyadenylic acid copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a deoxyadenylic acid polymer is also included in the deoxyadenylic acid polymer.
  • the deoxyadenylic acid copolymer is a polymer having a structural unit derived from dAMP and a structural unit derived from a nucleotide other than dAMP.
  • Nucleotides other than dAMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides. Examples of nucleotides other than dAMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, TMP, dTMP, UMP, dUMP, and combinations of two or more of these.
  • At least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, and AMP is preferable, and at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • Examples of the above-mentioned "derivative" and “salt” include those similar to those described in the above-mentioned "(1) Inosinic acid polymer".
  • the constituent units derived from dAMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 70 mol% or more, among all the constituent units. It is 75 mol% or more, 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. Further, the constituent unit derived from dAMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the dAMP-derived structural unit (which can also be expressed as base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, still more preferably 60 or more. Even more preferably, the number is 70 or more, and for example, 100 or more may be used. Further, the number of repetitions of the constituent unit derived from dAMP can be, for example, 10,000 or less, 8,000 or less, and 5,000 or less. The repetition may be continuous or intermittent, preferably continuous.
  • the deoxythymidylate polymer is used in the sense of including a deoxythymidylate homopolymer (polydeoxythymidylate), a deoxythymidylate copolymer, derivatives thereof, and salts thereof.
  • the deoxythymidylate polymer also includes a double-stranded nucleic acid polymer in which at least one of the double strands is a deoxythymidylate polymer (for example, polydeoxythymidylate).
  • the deoxythymidylate copolymer is a polymer having a constituent unit derived from dTMP and a constituent unit derived from a nucleotide other than dTMP.
  • Nucleotides other than dTMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than dTMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, UMP, dUMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and it is selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • At least one selected from the group consisting of IMP and dIMP is more preferable.
  • Examples of the above-mentioned “derivative” and “salt” include those similar to those described in the above-mentioned "(1) Inosinic acid polymer”.
  • the constituent units derived from dTMP are, for example, more than 50 mol%, preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 70 mol% or more, and more preferably 70 mol% or more, among all the constituent units.
  • the constituent unit derived from dTMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the dTMP-derived structural unit (which can also be expressed as base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, preferably 60 or more. More preferably, it may be 70 or more.
  • the number of repetitions of the constituent unit derived from dTMP can be, for example, 10,000 or less, 8,000 or less, 5000 or less, 3000 or less, and 1000 or less. The repetition may be continuous or intermittent, preferably continuous.
  • Deoxyuridylic acid polymer is used in the sense of including deoxyuridyllic acid homopolymer (polydeoxyuridic acid), deoxyuridic acid copolymer, derivatives thereof, and salts thereof.
  • a double-stranded nucleic acid polymer in which at least one of the double strands is a deoxyuridyl acid polymer (for example, polydeoxyuridic acid) is also included in the deoxyuridyl acid polymer.
  • the deoxyuridylic acid copolymer is a polymer having a constituent unit derived from dUMP and a constituent unit derived from a nucleotide other than dUMP.
  • Nucleotides other than dUMP are not particularly limited and may be ribonucleotides or deoxyribonucleotides.
  • Examples of nucleotides other than dUMP include IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, dAMP, TMP, dTMP, UMP, and combinations of two or more of these. Of these, at least one selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, AMP, and dAMP is preferable, and it is selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, and dGMP.
  • At least one selected from the group consisting of IMP and dIMP is more preferable.
  • 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 70 mol% or more, among all the constituent units. Is 75 mol% or more, 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.
  • the constituent unit derived from dUMP may be 100 mol% (that is, homopolymer), or may be, for example, 99.9 mol% or less, or 99 mol% or less, based on all the constituent units.
  • the number of repetitions of the dUMP-derived structural unit (which can also be expressed as a base length or the like) is, for example, 30 or more, preferably 40 or more, more preferably 50 or more, preferably 60. As mentioned above, more preferably 70 or more, for example, 100 or more, 150 or more may be used. Further, the number of repetitions of the constituent unit derived from dUMP can be, for example, 10000 or less, 8000 or less, 5000 or less, 3000 or less, 1000 or less. The repetition may be continuous or intermittent, preferably continuous.
  • the nucleic acid polymer comprises a nucleotide-derived structural unit selected from the group consisting of IMP, dIMP, CMP, dCMP, GMP, dGMP, derivatives thereof, and salts thereof, among all the structural units.
  • IMP IMP
  • CMP CMP
  • dCMP CMP
  • GMP dGMP
  • salts thereof among all the structural units.
  • 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.
  • the nucleic acid polymer comprises, for example, 60 mol% or more of nucleotide-derived constituent units selected from the group consisting of IMP, dIMP, derivatives thereof, and salts thereof, among all the constituent units. It is 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 90 mol% or more.
  • nucleic acid polymer containing 95 mol% or more.
  • the upper limit is not particularly limited, but for example, these constituent units may be 100 mol%, 99.9 mol% or less, or 99 mol% or less of the nucleic acid polymer in all the constituent units. ..
  • the nucleic acid polymer is polyinosic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polythimidic acid, polyuridylic acid, polydeoxyinosic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid. , Polydeoxythymidylic acid, polydeoxyuridylic acid, and at least one selected from the group consisting of salts thereof.
  • the nucleic acid polymer is polyinosinic acid, polycitidilic acid, polyguanylic acid, polyadenylic acid, polydeoxyinosinic acid, polydeoxycitidilic acid, polydeoxyguanyl acid, polydeoxyadenylic acid, and salts thereof. It is at least one selected from the group consisting of. In a more preferred embodiment, the nucleic acid polymer is at least selected from the group consisting of polyinosinic acid, polycitidilic acid, polyguanylic acid, polydeoxyinosinic acid, polydeoxycitidilic acid, polydeoxyguanylic acid, and salts thereof. It is a kind. 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 has a total length of 25 to 15,000 bases, preferably 30,000 to 10,000 bases, more preferably 40 to 9000 bases, and more. It is preferable to include the above-mentioned nucleic acid polymer having a total length of 50 to 8000 bases, and may be, for example, 100 to 5000 bases. For example, the content of the nucleic acid polymer having a total length of 25 to 15,000 bases, 30 to 10000 bases, 40 to 9000 bases, 50 to 8000 bases, or 100 to 5000 bases is the entire nucleic acid polymer, respectively.
  • the nucleic acid polymer may be a mixture of nucleic acid polymers having various base lengths, for example, a mixture of nucleic acid polymers having a substantially distributed range of 25 to 15000 base lengths. It is preferably a mixture of nucleic acid polymers that is substantially distributed in the range of 30 to 10,000 base lengths, and more preferably a mixture of nucleic acid polymers that is substantially distributed in the range of 40 to 9000 base lengths.
  • nucleic acid polymers such that they are substantially distributed in the range of 50 to 8000 bases, for example, a mixture of nucleic acid polymers that are substantially distributed in the range of 100 to 5000 bases is used. You can also do it.
  • substantially distributed in the range of base lengths A to B means that nucleic acid polymers having base lengths A to B account for, for example, 70 mol% or more of the total nucleic acid polymer.
  • the nucleic acid polymer may be, for example, a mixture of nucleic acid polymers having a distribution having a peak (or average value or mode) around 30 to 3000 base lengths, or a peak around 50 to 1500 base lengths (or a peak value or mode).
  • it may be a mixture of nucleic acid polymers having a distribution having an average value or a mode), or a distribution having a peak (or an average value or a mode) in the vicinity of 60 to 1000 base lengths. It is preferably a mixture of nucleic acid polymers, and a mixture of nucleic acid polymers showing a distribution having a peak (or average value or mode) in the vicinity of 70 to 800 base lengths can also be used. “Nearby” means that the lower 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, still more preferably 60 bases or more, still more preferably 70 bases or more, for example, 100 bases or more, 150 bases or more. It may be. As described above, the total length of the nucleic acid polymer is preferably longer than the base length designed as a probe or primer. Further, the total length of the nucleic acid polymer may be, for example, 10000 bases or less, further 8000 bases or less, or 5000 bases or less, for example.
  • the nucleic acid polymer can be synthesized by any method that can be used for the synthesis of oligonucleotides, for example, a phosphoric acid triester method, an H-phosphonate method, a thiophosphonate method, or the like.
  • natural products may be used, and commercially available products can also be preferably used.
  • a commercially available product for example, a commercially available nucleic acid polymer such as a potassium polyinosate (manufactured by Sigma), a potassium polyadenylate (manufactured by Sigma), or a potassium polyinosate (manufactured by Santa Cruz) can be used.
  • the one-step RT-PCR reaction solution used in the present invention contains, for example, the above-mentioned nucleic acid polymer, template RNA, primer, deoxyribonucleotide, and DNA polymerase having reverse transcription activity. Other components such as reverse transcriptase may be optionally included. Therefore, the composition for use in the one-step RT-PCR reaction of the present invention may optionally contain the above-mentioned components.
  • the concentration of the nucleic acid polymer is, for example, 0.1 ng / ⁇ L or more, more preferably 1 ng, from the viewpoint of more effectively suppressing non-specific nucleic acid amplification in the one-step RT-PCR reaction.
  • the concentration of the nucleic acid polymer may be, for example, 100,000 ng / ⁇ L or less, preferably 5000 ng / ⁇ L or less, more preferably 3000 ng / ⁇ L or less, 2000 ng / ⁇ L or less, 1500 ng / ⁇ L or less, 1000 ng / ⁇ L or less.
  • the composition for use in the 1-step RT-PCR reaction of the present invention preferably contains the nucleic acid polymer in an amount prepared so that the final concentration in the 1-step RT-PCR reaction solution becomes the above concentration. ..
  • RNA examples include biological samples such as tissues, body fluids or secretions (for example, biological tissues, body fluids or secretions that may contain pathogenic microorganisms such as viruses), environmental samples, and cells (for example, cultured cells) collected from the living body. It can be RNA derived from, for example, a biological sample, an environmental sample, or an RNA extracted from a cell, or an RNA further purified after the extraction treatment (for example, ethanol precipitation, column purification, etc.). Purified RNA) or the like processed by any purification means known in the field may be used, and may be any RNA. As one form, when RNA is extracted from a tissue, the site of the tissue is not particularly limited and may be any tissue.
  • the method for extracting RNA from a tissue usually includes a step of isolating cells by a cell strainer, trypsin treatment, or the like, but the method is not particularly limited.
  • RNA when RNA is extracted from a cell, it is not particularly limited and may be any kind of cell.
  • the number of cells can be appropriately adjusted by a cell sorter.
  • RNA extracted from a small number of cells eg, 1-100, preferably 1-10, more preferably 1 or 2, more preferably 1
  • a method for extracting RNA from a cell usually comprises the step of lysing the cell using a cytolytic composition containing a cytolytic agent.
  • the cytolytic agent include surfactants and chaotropic agents.
  • Surfactants include anionic surfactants (eg, sodium dodecyl sulfate, sodium sulfonic acid, sodium deoxycholate), cationic surfactants (eg, cetyltrimethylammonium bromide), nonionic surfactants (eg, cetyltrimethylammonium bromide). , Octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate), and amphoteric surfactants (eg, 3-[(3-colamidopropyl) dimethylammonio]. -1-Propane sulfonic acid) is included.
  • anionic surfactants eg, sodium dodecyl sulfate, sodium sulfonic acid, sodium deoxycholate
  • cationic surfactants eg, cetyltrimethylammonium bromide
  • nonionic surfactants eg, cetyltrimethylammonium bromide
  • the chaotropic agent examples include lithium salts such as urea and lithium perchlorate.
  • the cytolytic agent is usually dissolved in water (preferably nuclease-free water) and used in the form of an aqueous solution.
  • the composition for cytolysis may contain a protease (for example, protease K), an RNase inhibitor, and a combination of two or more of these.
  • the cytolytic composition may or may not contain the above nucleic acid polymer.
  • RNA extracted from cells using the composition for cell lysis containing the above nucleic acid polymer is used as a template RNA, the above nucleic acid polymer does not need to be added separately to the composition for use in the one-step RT-PCR reaction. May be good.
  • the biological samples include, for example, feces (excretion, rectal stool), urine, vomitus, saliva, sputum, pharyngeal swab, nasal swab, runny nose, and blood (whole). Blood), plasma, serum and the like can be mentioned, but the present invention is not limited, and it can be used for all substances derived from a living body, and can be appropriately selected depending on the purpose.
  • a biological sample such as a fecal sample, a blood sample, or a pharyngeal swab sample is used as the biological sample.
  • the target template RNA may be RNA derived from a living body, or RNA derived from a microorganism or virus contained in a biological sample.
  • the sample may be directly subjected to detection, or the sample is suspended 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. It may be a sample.
  • the buffer solution is not particularly limited, and examples thereof include Hanks buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and tricine buffer solution.
  • the present invention can be practiced to amplify nucleic acids from RNA in environmental samples such as wipe test samples.
  • the present invention can be used for detecting target RNA from a wiping test sample, a soil sample, a sewage sample, etc. as described above, but is not particularly limited, and any sample derived from the environment can be used. Wiping inspection is useful for elucidating the route of contamination by viruses and bacteria and for understanding the status of contamination such as the facility environment.
  • the wiping test is not particularly limited, but is a sample obtained by wiping the relevant section or equipment with a cotton swab or the like, eluting it in water or a buffer solution, and concentrating it with polyethylene glycol (PEG) precipitate or the like. ..
  • Specific examples of the wiping test procedure include "improvement of the norovirus test method for wiped samples" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). There is no particular limitation, and methods similar to this are widely included.
  • wipes examples include kitchen utensils such as cutting boards, kitchen knives, towels, and tableware, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilets, washbasins, handrails, living rooms and other facilities. Although it is not a wiping test, it can also be applied to a concentrated sample of a sewage sample as an environmental test.
  • RNA virus When the test target contained in the biological sample is an RNA virus, it is not particularly limited, and it may be an RNA virus having an envelope derived from a lipid bilayer or a non-enveloped RNA virus. good. RNA viruses having envelopes include flavivirus family viruses (eg, hepatitis C virus, Japanese encephalitis virus, decavirus); Togavirus family virus (eg, ruin virus, tikungunia virus); coronavirus family virus (eg, SARS).
  • flavivirus family viruses eg, hepatitis C virus, Japanese encephalitis virus, decavirus
  • Togavirus family virus eg, ruin virus, tikungunia virus
  • coronavirus family virus eg, SARS
  • Coronavirus SARS-CoV-1
  • MERS coronavirus
  • COVID-19 coronavirus SARS-CoV-2
  • Orthomixoviridae virus eg, influenza virus (influenza virus type A, influenza virus type B, influenza) Virus C type, etc.)
  • Rabdoviridae virus eg, mad dog disease virus
  • Bunyavirus family virus eg, Crimea congo fever virus
  • Paramyxoviridae virus eg, measles virus, human RS virus
  • Phyllovirus family Examples include viruses (eg, Ebola virus).
  • the enveloped RNA virus is to be tested, from the viewpoint that the present invention is more effective, it is preferably a coronaviridae virus or an orthomyxoviridae virus, and more preferably SARS-CoV-1, SARS-CoV-. 2. It can be influenza virus type A or influenza virus type B.
  • Non-enveloped RNA viruses include astroviridae virus (eg, astrovirus); caliciviridae virus (eg, sapovirus, norovirus); picornavirus family virus (eg, hepatitis A virus, echovirus, enterovirus, coxsackie virus). , Poliovirus, Rhinovirus); Hepevirus family virus (eg, hepatitis E virus); Leovirus family virus (eg, Rotavirus) and the like.
  • astroviridae virus eg, astrovirus
  • caliciviridae virus eg, sapovirus, norovirus
  • picornavirus family virus eg, hepatitis A virus, echovirus, enterovirus, coxsackie virus.
  • Poliovirus, Rhinovirus Poliovirus, Rhinovirus
  • Hepevirus family virus eg, hepatitis E virus
  • Leovirus family virus eg, Rotavirus
  • RNA virus When a non-enveloped RNA virus is to be tested, it may be a Caliciviridae virus or a Leovirus family virus, and more preferably a norovirus, a sapovirus, or a rotavirus from the viewpoint that the present invention is more effective. ..
  • the present invention can be carried out to amplify nucleic acid from any of the above RNA viruses, but in one embodiment, from the viewpoint that the effects of the present invention can be more reliably obtained, preferably a non-enveloped virus. It can be performed to detect RNA, more preferably Caliciviridae virus RNA, and even more preferably norovirus RNA. As shown in the results of the test examples described later, according to the present invention, norovirus G1 can effectively suppress non-specific reactions even by 1-step RT-PCR from a low copy of norovirus RNA. It is possible to detect genes and G2 genes with high sensitivity.
  • the concentration of the template RNA added to the 1-step RT-PCR reaction solution is, for example, 1 to 10000 pg / ⁇ L, preferably 10 to 1000 pg / ⁇ L, and more preferably 1 to 100 pg / ⁇ L. Not limited.
  • a heat-resistant DNA polymerase belonging to Family A and having reverse transcription activity is used as the DNA polymerase.
  • the DNA polymerase having reverse transcription activity is a DNA polymerase having both the ability to convert RNA into cDNA and the ability to amplify DNA, and by using such a DNA polymerase, one step of a one-enzyme system. It may be possible to carry out an RT-PCR reaction.
  • the heat resistance means that the enzyme activity does not decrease by more than half even if the heat treatment is performed at 70 ° C. for 1 minute or more.
  • DNA polymerases used for PCR DNA polymerases belonging to family A derived from thermophiles (also called polI type) and DNA polymerases belonging to family B derived from hyperthermophilic archaea (also called ⁇ type) have been conventionally used. Etc. are known.
  • the heat-resistant polymerase belonging to Family A is not particularly limited, but for example, a DNA polymerase (Tth polymerase) derived from Thermus thermophilus HB8, Thermus sp. Z05-derived DNA polymerase (Hawk Z05 polymerase), Thermotoga maritima-derived DNA polymerase (Tma polymerase), Bacillus caldotenex-derived DNA polymerase (Bca polymerase), Bacillus theatermorphis-derived DNA polymerase (Bst polymerase), etc. It may be a variant in which the reverse transcription activity and the heat-resistant DNA polymerase activity are not lost.
  • Tth polymerase derived from Thermus thermophilus HB8, Thermus sp. Z05-derived DNA polymerase
  • Tma polymerase Thermotoga maritima-derived DNA polymerase
  • Bca polymerase Bacillus caldotenex-derived DNA polymerase
  • Bacillus theatermorphis-derived DNA polymerase Bacillus
  • a DNA polymerase having reverse transcription activity selected from the group consisting of Tth, Z05 and variants thereof can be mentioned.
  • Tth polymerase preferably at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and variants thereof, and by using these, even when a sample containing a large amount of insoluble substance is used. , It is possible to detect RNA virus with even 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 for the present invention are shown in the sequence listing.
  • a mutant DNA polymerase (DNA polymerase variant) in which some amino acids are modified based on these amino acid sequences without losing the effect can also be preferably used.
  • the variant of the heat-resistant DNA polymerase having reverse transcription activity is, for example, 85% or more, preferably 90% or more, more preferably 95% or more, based on the amino acid sequence of the wild-type DNA polymerase from which it is derived. % Or more, more preferably 98% or more, particularly preferably 99% or more, and having an activity of converting RNA into cDNA and an activity of amplifying DNA like wild-type DNA polymerase.
  • a method for calculating the identity of the amino acid sequence any means known in the art can be used. For example, it can be calculated using a commercially available analysis tool or an analysis tool available through a telecommunication line (Internet).
  • the National Center for Biotechnology Information (NCBI) homology algorithm BLAST Basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic local alignment search tool
  • It may be a polypeptide consisting of an amino acid sequence (also referred to as "mutation"), and may have an activity of converting RNA into DNA and an activity of amplifying DNA as in the case of wild-type DNA polymerase.
  • the number of 1 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 the number is not particularly limited.
  • the total amount of the heat-resistant DNA polymerase (preferably a heat-resistant DNA polymerase having reverse transcriptase activity belonging to Family A) contained in the one-step RT-PCR reaction solution exerts the effect of the present invention.
  • the limitation is not particularly limited, but as an example, it may be at least 2.0 ng / ⁇ L or more, preferably 3.0 ng / ⁇ L or more, and more preferably 4.2 ng / ⁇ L or more.
  • the upper limit of the total amount of the thermostable DNA polymerase contained in the 1-step RT-PCR reaction solution is not particularly limited, but as an example, it may be 20 ng / ⁇ L or less, and may be 10 ng / ⁇ L or less, for example, 6 ng.
  • the composition for use in the 1-step RT-PCR reaction of the present invention is the heat-resistant DNA polymerase (preferably) in an amount prepared so that the final concentration in the 1-step RT-PCR reaction solution is the above concentration.
  • a heat-resistant DNA polymerase having reverse transcriptase activity belonging to Family A) is preferably contained.
  • the amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be estimated from the Safety Data Sheet (SDS). When a protein such as BSA is contained, it is desirable to calculate by the latter method.
  • the method for suppressing non-specific nucleic acid amplification and amplifying the template RNA in the sample is characterized in that at least the following steps are included.
  • (1) A step of preparing a mixed solution by mixing a sample containing a template RNA with a 1-step RT-PCR reaction solution containing the nucleic acid polymer; (2) A step of carrying out a one-step RT-PCR reaction after sealing the reaction vessel.
  • the step (1) it is preferable to include a thermostable DNA polymerase having reverse transcription activity, which may enable a one-step RT-PCR reaction in a one-enzyme system.
  • the steps (1) and (2) are preferably performed in the same container. That is, it is preferable not to transfer all or a part of the mixed solution to another container between the steps (1) and (2). Further, in the 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 the step (2) is 1. Reverse transcription reaction, 2. It consists of two steps of 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 of 1 is determined by the reverse transcription activity of the thermostable DNA polymerase and the Tm values of the primers and probes, and may be at least 25 ° C. or higher. More preferably, it is 37 ° C. or higher.
  • [1] DNA denaturation by heat treatment dissociation from double-stranded DNA to single-stranded DNA
  • [2] annealing of primers to template single-stranded DNA [3] the above using DNA polymerase.
  • the thermal cycler used for the RT-PCR reaction has a total extension time of steps [2] and [3] of 60 seconds or less, preferably 45 seconds or less. More preferably, it is desirable to set a measurement program of 30 seconds or less.
  • the “PCR extension time” refers to the set temperature in the thermal cycler.
  • the 1-step RT-PCR reaction solution used in the present invention is introduced into a thermally unstable block group DNA polymerase in combination with an anti-DNA polymerase antibody or by chemical modification in order to enhance the effect of suppressing non-specific reaction.
  • the 1-step RT-PCR reaction solution used in the present invention includes a heat-resistant DNA polymerase, a nucleic acid polymer, a buffer, an appropriate salt, a magnesium salt or a manganese salt, a deoxynucleotide triphosphate, and a template RNA to be detected.
  • a primer pair corresponding to the detection target region and, if necessary, an additive may be contained. Therefore, the composition for use in the one-step RT-PCR reaction of the present invention may optionally contain the above-mentioned components.
  • the buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, and Bicine.
  • the pH was adjusted to 6 to 9, more preferably pH 7 to 9, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like.
  • the concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM.
  • a salt solution is added in order to make the ionic conditions suitable for the reaction.
  • the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like.
  • dNTP used as a substrate in the present invention
  • dATP, dCTP, dGTP, and dTTP are added at 0.1 to 0.5 mM, respectively, and most commonly, about 0.2 mM is added.
  • Prophylactic measures against cross-contamination may be taken by using dUTP as an alternative and / or as part of dTTP.
  • UNG Uracil-N-glycosylase
  • the monoenzyme-based 1-step RT-PCT reaction solution contains a divalent cation.
  • the divalent cation is not particularly limited, and examples thereof include magnesium ion, manganese ion, calcium ion, copper ion, iron ion, nickel ion, and zinc ion.
  • the divalent cation preferably contains magnesium ion and manganese ion.
  • magnesium ions, manganese ions, or the like are added to the one-enzyme system 1-step RT-PCR reaction solution, magnesium or manganese may be added, or salts thereof may be added.
  • magnesium or a salt thereof examples include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate and the like, and examples of manganese or a salt thereof include manganese, manganese chloride, manganese sulfate, manganese acetate and the like.
  • Such magnesium, manganese, or salts thereof are preferably added to the RT-PCR reaction solution in an amount of about 1 to 10 mM.
  • manganese or a salt thereof is preferably contained from the viewpoint that stable and high sensitivity can be easily obtained.
  • the 1-step RT-PCR reaction solution preferably contains 1 mM or more of manganese or a salt thereof, preferably 1.5 mM or more of manganese or a salt thereof, and 2.0 mM or more of manganese or a salt thereof. More preferably, it contains salt.
  • albumin a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid
  • albumin may contain at least one selected from the group consisting of (eg, bovine serum albumin, etc.), sericin, BPF, glycerol, glycol, gelatin (eg, fish gelatin, pig gelatin, etc.) and surfactants.
  • albumin and / or gelatin is preferably contained, both albumin and gelatin are preferably contained, and bovine serum albumin and fish gelatin and / or porcine gelatin are particularly preferable.
  • the one-step RT-PCR reaction solution contains bovine serum albumin (also abbreviated as BSA), its concentration is not particularly limited, but for example, it is preferably at least 0.5 mg / mg with respect to the entire RT-PCR reaction solution. It is ml or more, more preferably at least 1 mg / ml or more. In a sample containing a large amount of impurities, the concentration of bovine serum albumin is preferably 2 mg / ml or more, more preferably 3 mg / ml or more, and good detection is possible. The upper limit is not particularly limited, but as an example, it can be 10 mg / ml or less.
  • BSA bovine serum albumin
  • the one-step RT-PCR reaction solution contains gelatin (for example, fish gelatin, pig gelatin), its concentration is not particularly limited, but for example, it is preferably at least 0.5 mg with respect to the entire RT-PCR reaction solution. / Ml or more, more preferably at least 1 mg / ml or more, still more preferably 5 mg / ml or more.
  • the gelatin concentration is preferably 7.5 mg / ml or more, more preferably 15 mg / ml or more, and good detection is possible.
  • the upper limit is not particularly limited, but as an example, it can be 30 mg / ml or less.
  • Examples of the surfactant contained in the 1-step RT-PCR reaction solution include Triton X-100, Triton X-114, Tween 20, Nonidet P40, Briji35, and Briji58. Examples thereof include SDS, CHAPS, CHASPO, and Emulgen 420, but the present invention is not particularly limited.
  • 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, still more preferably 0.005% or more, and good detection is achieved. Is possible.
  • the upper limit is not particularly limited, but as an example, it can be 0.1% or less.
  • betaine-like quaternary ammonium contained in the 1-step RT-PCR reaction solution examples include betaine (trimethylglycine) and carnitine, but are not particularly limited.
  • the betaine structure is a compound having both positive and negative charges that are stable in the molecule, and is known to exhibit surfactant-like properties and promote nucleic acid amplification of DNA polymerase.
  • the preferred betaine-like quaternary ammonium concentration is 0.1M to 2M, more preferably 0.2M to 1.2M.
  • Accelerators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single strand binding proteins (SSBs), T4 gene 32 proteins, tRNA, sulfur or acetamide-containing compounds, dimethylsulfommonium (DMSO), glycerol, ethylene.
  • Glycerol Propylene Glycol, Trimethylene Glycol, Formamide, Acetamide, Ectoin, Trehalose, Dextran, Polyvinylpyrrolidone (PVP), Tetramethylammonium Chloride (TMC), Tetramethylammonium Hydroxide (TMAH), Tetramethylammonium Acetate (TMAA), Examples thereof include, but are not limited to, polyethylene glycol.
  • EGTA Ethyleneglycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid
  • BAPTA a chelating agent
  • primer pair used in the present invention examples include a pair of two types of primers in which one primer is complementary to the DNA extension product of the other primer. Further, as another embodiment, so-called multiplex PCR in which two or more pairs of the above primers are contained can be mentioned.
  • degenerate primers may be included if the target nucleic acid consists of subtypes.
  • the primer concentration to be detected is not particularly limited, but the concentration of the forward primer is 0.1 ⁇ M or more and 3 ⁇ M or less and the concentration of the reverse primer is 0.1 ⁇ M or more and 3 ⁇ M with respect to the entire RT-PCR reaction solution. The following is preferable. More preferably, the concentration of the forward primer is 0.1 ⁇ M or more and 2 ⁇ M or less, and the concentration of the reverse primer is 0.5 ⁇ M or more and 2 ⁇ M or less.
  • Another aspect of the present invention is a detection method further comprising at least one labeled hybridization probe or double-stranded DNA-bound fluorescent compound.
  • Examples of the double-stranded DNA-bound fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), and EvaGreen (registered trademark; Biotium). , LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science) and the like.
  • hybridization probe used in the present invention examples include TaqMan hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972). , US Pat. No. 5,804,375), Molecular Beacon (US Pat. No. 5,118,801), FRET Hybridization Probe (International Publication No. 97/46707, International Publication No. 97/46712) , International Publication No. 97/46714 Pamphlet).
  • the concentration of the fluorescently labeled probe is preferably 0.01 ⁇ M or more and 1.0 ⁇ M or less. More preferably, it is 0.013 ⁇ M or more and 0.75 ⁇ M or less, and further preferably 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • the method for determining whether or not the amplification of non-specific nucleic acid is suppressed is not limited, but the amplification product after RT-PCR is subjected to electrophoresis and the base of the template RNA is subjected to electrophoresis. It can be confirmed by comparing with the band estimated from the number. In electrophoresis, the amount of non-specific nucleic acid amplification can be qualitatively determined by the strength of the electrophoresis band.
  • Another mode is a detection method containing a double-stranded DNA-bound fluorescent compound.
  • the melting curve analysis in this method it is possible to distinguish between the amplification of the target nucleic acid and the non-specific nucleic acid amplification from the difference in the Tm value of the amplification product. By comparing the peak heights on the melting curve, it is possible to determine the amount of non-specific nucleic acid amplification. Further, as yet another form, it can be confirmed by a detection method using a hybridization probe (for example, TaqMan hydrolysis probe).
  • a hybridization probe for example, TaqMan hydrolysis probe.
  • the number of PCR reaction cycles in which the fluorescence intensity improves according to the amount of nucleic acid amplification and reaches the threshold fluorescence intensity is called a Ct value. Increased non-specific nucleic acid amplification causes a decrease in reached fluorescence intensity. Therefore, the amount of non-specific nucleic acid amplification can be determined by comparing the reached fluorescence intensities.
  • compositions for suppressing non-specific nucleic acid amplification from a template RNA in a sample and amplifying nucleic acid by 1-step RT-PCR are a composition for suppressing non-specific nucleic acid amplification from a template RNA in a sample and amplifying nucleic acid by 1-step RT-PCR.
  • a composition for use in one-step RT-PCR of a one-enzyme system characterized in that it contains a particular nucleic acid polymer and a thermostable DNA polymerase having reverse transcription activity belonging to Family A, in a particular preferred particular embodiment.
  • the present invention may also be provided in the form of a kit containing these compositions for use in one-step RT-PCR in which non-specific nucleic acid amplification is suppressed.
  • kits of the present invention may be attached, for example, with instructions for use explaining the method of use of the present invention.
  • the kit of the present invention comprises a specific nucleic acid polymer, a thermostable DNA polymerase having reverse transcription activity belonging to Family A, and other components enclosed in the same container or in separate containers, for example. It can be packaged in a single package and provided in a manner that includes information on how to use the kit.
  • Test example 1 Examination of 1-enzyme system 1-step RT-PCR when potassium polyinosinate (inosinic acid polymer) was added 1 (1) Reaction solution Using the reaction solution having the composition shown below as the basic composition, norovirus RNA in the reaction solution was detected by 1-enzyme system 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • the liquid was prepared.
  • Norovirus RNA prepared at 1250 to 25 copies / ⁇ L was added in 1 ⁇ L increments, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad.
  • the fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • Test example 2 Examination of 1-enzyme system 1-step RT-PCR when potassium polyinosinate (inosinic acid polymer) was added 2 (1) Reaction solution Using the reaction solution having the composition shown below as the basic composition, norovirus RNA in the reaction solution was detected by 1-enzyme system 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • RT-PCR reaction solution was prepared so as to be.
  • Norovirus RNA prepared at 1250 to 25 copies / ⁇ L was added in 1 ⁇ L increments, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • Test example 3 Examination of 1-enzyme system 1-step RT-PCR when potassium polycitidilate (polymer citidilate) and potassium polyadenylate (polymer adenylate) were added (1) Reaction solution The reaction solution having the composition shown below was used as the basic composition. Norovirus RNA in the reaction solution was detected by 1-enzyme system 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • Potassium * (manufactured by Sigma, an enzyme synthetic product) was added so that the final concentration was 0 ng / ⁇ L and 100 ng / ⁇ L, and an RT-PCR reaction solution was prepared so that the final liquid volume was 49 ⁇ L.
  • Norovirus RNA prepared to have a copy number of 1250 copies / ⁇ L was added by 1 ⁇ L, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles.
  • the addition of potassium polyadenylate did not have a large inhibitory effect on G1 gene detection, but in G2 gene detection, an increase in the reached relative fluorescence intensity was confirmed at the time of addition, and non-specific nucleic acid amplification was suppressed. It was confirmed that.
  • the results when potassium polycitidilate is added are shown in FIGS. 12 to 13. In potassium polycitidilate, an increase in the reached relative fluorescence intensity was confirmed at 200 to 300 ng / ⁇ L for both the G1 and G2 genes, and it was confirmed that non-specific nucleic acid amplification was suppressed.
  • Test example 4 Examination of 1-enzyme system 1-step RT-PCR when polydeoxythymidylate (thymidylate polymer) and potassium polyguanylate (guanylate polymer) are added (1) Reaction solution The reaction solution having the composition shown below is used as the basic composition. Norovirus RNA in the reaction solution was detected in 1-enzyme system 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • Norovirus RNA prepared to have a copy number of 1250 copies / ⁇ L was added by 1 ⁇ L, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • Test example 5 Examination of 1-enzyme system 1-step RT-PCR when polyinosic acid-sodium polycitidilate (double-chain citidilate polymer) was added (1) Reaction solution The reaction solution having the composition shown below was used as the basic composition, and 1 Norovirus RNA in the reaction solution was detected by enzyme-based 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • the RT-PCR reaction solution was prepared so as to have a concentration of 49 ⁇ L.
  • Norovirus RNA prepared to have a copy number of 1250 copies / ⁇ L was added by 1 ⁇ L, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • polyinosic acid-sodium polycitidirate (Poly (I: C)) has peak base lengths of about 100 bases and about 150 bases, and is about 50 to 800, as shown in FIG. It was found to contain base-length polyinosic acid and polycitidilic acid (reference value).
  • Poly (I: C) has peak base lengths of about 100 bases and about 150 bases, and is about 50 to 800, as shown in FIG. It was found to contain base-length polyinosic acid and polycitidilic acid (reference value).
  • Test example 6 Examination of 1-enzyme system 1-step RT-PCR when potassium polyinosinate (inosinic acid polymer) was added (SARS-CoV-2 RNA detection) (1) Reaction solution Using the reaction solution having the composition shown below as the basic composition, SARS-CoV-2 RNA (region 1 and region 2 of the N gene) in the reaction solution was detected by 1-enzyme system 1-step RT-PCR. bottom.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • the RT-PCR reaction solution was prepared so as to be.
  • SARS-CoV-2 RNA manufactured by Twist Bioscience
  • SARS-CoV-2 RNA prepared at 1250 to 25 copies / ⁇ L was added in 1 ⁇ L increments, and RT-PCR was performed as a 50 ⁇ L reaction system.
  • This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad.
  • the fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • Test example 7 Examination of 1-enzyme system 1-step RT-PCR when potassium polyinosinate (inosinic acid polymer) was added (influenza virus RNA detection) (1) Reaction solution Using the reaction solution having the composition shown below as the basic composition, influenza virus type A or B RNA in the reaction solution was detected by 1-enzyme system 1-step RT-PCR.
  • the rTth DNA polymerase used in this test example is a thermostable DNA polymerase belonging to Family A and having reverse transcription activity.
  • the RT-PCR reaction solution was prepared so as to be. 1 ⁇ L of influenza virus type A or B RNA (manufactured by Twist Bioscience) prepared to 1250 to 25 copies / ⁇ L was added, and RT-PCR was performed as a 50 ⁇ L reaction system. This was subjected to a real-time PCR reaction in the following temperature cycle using CFX96WELL DEEP manufactured by BioRad. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles. Next, the reached relative fluorescence intensity calculated by BioRad's CFX Manager or CFX Maestro software was used as the measured value.
  • the present invention is suitably used in molecular biology research, and in tests for the purpose of clinical tests, food hygiene management, and the like.

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Abstract

La présente invention a pour but de fournir un moyen pour effectuer spécifiquement une amplification d'ADN à partir d'ARN matriciel par suppression de l'amplification d'acide nucléique non spécifique par l'intermédiaire d'un procédé de RT-PCR en une étape. La présente invention fournit un procédé pour supprimer l'amplification non spécifique des acides nucléiques en utilisant une solution de réaction RT-PCR en une étape contenant au moins un polymère d'acide nucléique choisi dans le groupe constitué par un polymère d'acide inosinique, un polymère d'acide cytidylique, un polymère d'acide guanylique, un polymère d'acide adénylique, un polymère d'acide thymidylique, un polymère d'acide uridylique, un polymère d'acide désoxyinosinique, un polymère d'acide désoxycytidylique, un polymère d'acide désoxyguanylique, un polymère d'acide désoxyadénylique, un polymère d'acide désoxythymidylique et un polymère d'acide désoxyuridylique.
PCT/JP2021/006071 2020-03-02 2021-02-18 Procédé pour supprimer l'amplification non spécifique des acides nucléiques WO2021177041A1 (fr)

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JP2011504754A (ja) * 2007-11-27 2011-02-17 バイオ−ラッド ラボラトリーズ，インコーポレイティド １ステップｒｔ−ｐｃｒの阻害低減
JP4777497B2 (ja) * 1999-05-03 2011-09-21 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング リボ核酸類からの核酸類の生成及び増幅
JP2020018295A (ja) * 2018-07-24 2020-02-06 東洋紡株式会社 改良されたウイルス検出方法
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