WO2022210122A1 - Ensemble d'oligonucléotides pour détecter une pluralité de types de virus par pcr multiplex - Google Patents

Ensemble d'oligonucléotides pour détecter une pluralité de types de virus par pcr multiplex Download PDF

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WO2022210122A1
WO2022210122A1 PCT/JP2022/013261 JP2022013261W WO2022210122A1 WO 2022210122 A1 WO2022210122 A1 WO 2022210122A1 JP 2022013261 W JP2022013261 W JP 2022013261W WO 2022210122 A1 WO2022210122 A1 WO 2022210122A1
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seq
nucleic acid
nucleotide sequences
acid primers
primer
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健介 大地
千恵 川井
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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to a set of oligonucleotides capable of detecting nucleic acids derived from multiple viruses, including influenza A virus and influenza B virus, by nucleic acid amplification.
  • the method of the present invention allows highly sensitive detection of viruses in biological samples such as, for example, pharyngeal and nasal swabs, sputum and saliva.
  • the present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection, and the like.
  • Nucleic acid amplification is a technology that amplifies a few copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more. It is also widely used in microbiological examinations, etc.
  • a typical nucleic acid amplification method is PCR (Polymerase Chain Reaction).
  • PCR consists of (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) conversion of the primer using a DNA polymerase. This is a method of amplifying a target nucleic acid in a sample by repeating three steps of elongation as one cycle. In some cases, annealing and extension are performed at the same temperature in two steps.
  • RT Reverse Transcription
  • This RT-PCR consists of (1) 2-step RT-PCR in which RT and PCR are performed discontinuously, (2) RT and PCR in succession using a single enzyme 1-step RT -PCR, and (3) a two-enzyme one-step RT-PCR in which RT and PCR are successively performed using two enzymes, reverse transcriptase and DNA polymerase.
  • PCR and RT-PCR are widely used for genetic testing and virus testing.
  • Typical examples of virus tests include pathogenic RNA viruses such as influenza viruses and coronaviruses.
  • Influenza virus is a causative virus of respiratory infections that is prevalent in winter and causes symptoms such as fever.
  • Influenza viruses are classified into a plurality of types according to the types of nucleoprotein (NP) and matrix protein (M), of which types A and B are frequently seen as seasonal influenza. It is known that, due to the difference in antigenicity between influenza A virus and influenza B virus, infection with one virus usually does not produce antibodies against the other virus, and thus, infection with both viruses is possible.
  • NP nucleoprotein
  • M matrix protein
  • Coronaviruses are causative viruses that cause respiratory infections including colds, and about 10 to 35% of colds are said to be caused by coronaviruses. Mutant viruses are also known to occur, and rarely SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, novel coronavirus infectious disease (COVID-19) coronavirus (SARS) -nCOV-2, or SARS-CoV-2) are known to cause fatal serious respiratory diseases.
  • Non-Patent Document 1 One method of speeding up the identification of viruses that cause respiratory infections is to test for multiple types of viruses at the same time in a single test. In recent years, multiplex PCR targeting nucleic acids derived from multiple viruses has been used in genetic testing for respiratory infections (Non-Patent Document 1). Also known is a method of detecting a virus by contacting a sample containing a virus such as influenza virus with a solution containing a water-soluble organic solvent to extract nucleic acid derived from the virus and amplifying the nucleic acid (Patent Document 1).
  • RNA nucleic acids including RNA nucleic acids
  • multiple types of nucleic acid primer sets are included in one RT-PCR reaction system.
  • the reverse transcription reaction and the PCR reaction for multiple types of target nucleic acid RNA are carried out in one solution, the multiple types of primer sets contained in the reaction solution mutually affect the amplification reaction of each nucleic acid. It has been known.
  • the difference in the Tm value of each primer also affects the reaction system. As a result, non-specific amplification or fundamental amplification failure occurs in reverse transcription and PCR reactions, respectively.
  • Non-specific amplification is also caused in the absence of the target nucleic acid, and is known to be one of the causes of false positives in genetic testing. This is particularly a concern in the multiplex PCR method for detecting two or more target nucleic acids, in which the optimal nucleic acid amplification/detection conditions tend to differ and the reaction tends to be biased.
  • RNA samples such as pharyngeal and nasal swabs, sputum, and saliva are used for genetic testing by RT-PCR.
  • Biological samples contain many contaminants that inhibit RT-PCR and non-viral nucleic acids that cause non-specific amplification. Therefore, if the RT-PCR reaction is performed using such a biological sample containing contaminants as it is without isolating the nucleic acid, the reaction system becomes even more complicated.
  • influenza A virus, influenza B virus, and coronavirus have similar respiratory disease symptoms and are difficult to diagnose from symptoms.
  • coronavirus especially SARS-nCOV-2
  • pharyngeal and nasal swabs, saliva, fecal samples, swab environmental samples, etc. by one-step RT-PCR, detect these multiple viruses at the same time, and It is desired to develop a further useful method in which the occurrence is also suppressed.
  • the object of the present invention is to highly suppress the occurrence of non-specific reactions even in a multiplex one-step RT-PCR reaction containing contaminants derived from biological samples such as saliva, pharynx and nasal swabs.
  • An object of the present invention is to provide a method capable of detecting the presence or absence of target nucleic acids derived from a plurality of viruses, including influenza A virus and influenza B virus, contained in a sample with higher sensitivity.
  • the present inventors have found that by using a primer set composed of nucleic acid primers of specific oligonucleotides, the occurrence of non-specific reactions is highly suppressed, and non-specific amplification can be reduced, and the presence or absence of target nucleic acids derived from a plurality of viruses, including influenza A virus and influenza B virus, contained in a sample can be detected with higher sensitivity.
  • this oligonucleotide set it is possible to overcome the decrease in detection sensitivity caused by the introduction of contaminants, and the PCR reaction can be performed without prior isolation and purification of nucleic acids from a sample that may contain contaminants.
  • the inventors have found that the target nucleic acid can be detected only by performing the above, and arrived at the present invention.
  • a method for detecting the presence or absence of target nucleic acids derived from influenza A virus and influenza B virus in a sample comprising the following steps: (1) Any primer set selected from the following combinations of groups (I) and (II), combinations of groups (III) and (IV), or combinations of groups (V) and groups (VI) wherein the combination of primer sets comprises at least two pairs of primer sets, at least one pair each selected from each of the groups, step: (I) Primer set a and primer set b (II) Primer Set f and Primer Set g (III) primer set c (IV) Primer set e and primer set g (V) Primer set d (VI) Primer set e and primer set f [The primer sets a to g are as follows: (Primer set a): a set of nucleic acid primers consisting of the base sequences shown in SEQ ID NO: 31 and SEQ ID NO: 33, or a set of nucleic acid
  • PCR reaction solution further comprises at least one pair of primer sets for detecting the presence or absence of a target nucleic acid derived from SARS-CoV-2 coronavirus selected from the following group (VII):
  • a set of nucleic acid primers a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26, a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NO: 28 and SEQ ID NO: 29, or each nucleic acid in these sets
  • a set of nucleic acid primers comprising nucleotide sequences complementary to the nucleotide sequences of the primers.
  • the PCR reaction solution further contains at least one nucleic acid probe consisting of a nucleotide sequence represented by any of SEQ ID NO: 35, SEQ ID NO: 41, or SEQ ID NO: 47 or a nucleotide sequence complementary thereto. Item 4. The method according to any one of items 1 to 3.
  • the PCR reaction solution further contains at least one nucleic acid probe consisting of a nucleotide sequence represented by any of SEQ ID NO: 38, SEQ ID NO: 44, or SEQ ID NO: 50 or a nucleotide sequence complementary thereto. Item 5. The method according to any one of Items 1 to 4.
  • the PCR reaction solution further contains SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, or SEQ ID NO: Item 6.
  • the probe is a TaqMan probe.
  • the sample is at least one selected from the group consisting of a saliva sample, a sputum sample, a gargle, tears, a throat swab, a nasopharyngeal swab, a nasal swab, a stool sample, and a swab test sample. 10.
  • the sample is a suspension suspended in at least one selected from the group consisting of water, physiological saline, buffer, and sputazyme enzyme solution, or a centrifugal supernatant or concentrate thereof.
  • [Item 15] Item 13, wherein the contaminant-resistant DNA polymerase is at least one contaminant-resistant DNA polymerase selected from the group consisting of Tth, Hawk Z05, and variants thereof. Or the method according to 14.
  • [Claim 16] A DNA in which the mutant comprises an amino acid sequence exhibiting 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 51) or Hawk Z05 polymerase (SEQ ID NO: 52), and having contaminant resistance. Item 16. The method of Item 15, which is indicative of polymerase activity.
  • the mutant comprises an amino acid sequence having deletion, substitution and/or addition of one or several amino acids in the amino acid sequence of Tth polymerase (SEQ ID NO: 51) or Hawk Z05 polymerase (SEQ ID NO: 52). 17.
  • the method according to Item 15 or 16 which exhibits a DNA polymerase activity having contaminant resistance.
  • the reverse transcriptase is at least selected from the group consisting of reverse transcriptase derived from Moloney murine leukemia virus (MMLV), reverse transcriptase derived from avian myeloblastosis virus (AMV), and variants thereof Item 18.
  • MMLV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • variants thereof Item 18 The method according to any one of Items 13 to 17, wherein the reverse transcriptase is derived from 1 species.
  • a kit comprising a primer set for detecting the presence or absence of a target nucleic acid derived from influenza A virus and influenza B virus in a sample, the kit comprising a combination of the following group (I) and group (II) , a combination of Group (III) and Group (IV), or a combination of Group (V) and Group (VI), wherein said combination of primer sets is selected from each of said A kit comprising at least two pairs of primer sets, at least one pair each selected from the group: (I) Primer set a and primer set b (II) Primer Set f and Primer Set g (III) primer set c (IV) Primer set e and primer set g (V) Primer set d (VI) Primer set e and primer set f [The primer sets a to g are as follows: (Primer set a): a set of nucleic acid primers consisting of the base sequences shown in SEQ ID NO: 31 and SEQ ID NO: 33, or a set of
  • a set of nucleic acid primers a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26, a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NO: 28 and SEQ ID NO: 29, or each nucleic acid in these sets
  • a set of nucleic acid primers comprising nucleotide sequences complementary to the nucleotide sequences of the primers.
  • the probe is a TaqMan probe.
  • the sample is at least one selected from the group consisting of a saliva sample, a sputum sample, a gargle, tears, a pharyngeal swab, a nasopharyngeal swab, a nasal swab, a fecal sample, and a swab sample.
  • the kit according to any one of items 19 to 26.
  • the sample is a suspension suspended in at least one selected from the group consisting of water, physiological saline, buffer, and sputazyme enzyme solution, or a centrifugal supernatant or concentrate thereof. Item 28. The kit according to any one of Items 19-27.
  • [Item 29] The kit according to any one of Items 19 to 28, further comprising a DNA polymerase.
  • [Item 31] The kit according to any one of Items 19 to 30, wherein the contaminant-resistant DNA polymerase is a DNA polymerase belonging to Family A.
  • the contaminant-resistant DNA polymerase is at least one contaminant-resistant DNA polymerase selected from the group consisting of Tth, Hawk Z05, and variants thereof. or the kit according to 31.
  • [Claim 33] A DNA in which the mutant comprises an amino acid sequence exhibiting 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 51) or Hawk Z05 polymerase (SEQ ID NO: 52), and having contaminant resistance.
  • the kit of Paragraph 32 which exhibits polymerase activity.
  • the mutant consists of an amino acid sequence having deletion, substitution and/or addition of one or several amino acids in the amino acid sequence of Tth polymerase (SEQ ID NO: 51) or Hawk Z05 polymerase (SEQ ID NO: 52). 34.
  • the kit according to Item 32 or 33 which exhibits a DNA polymerase activity having contaminant resistance.
  • the reverse transcriptase is at least selected from the group consisting of reverse transcriptase derived from Moloney murine leukemia virus (MMLV), reverse transcriptase derived from avian myeloblastosis virus (AMV), and variants thereof Item 35.
  • MMLV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • the kit according to any one of Items 30 to 34, wherein the reverse transcriptase is derived from 1 species.
  • target nucleic acids derived from two or more viruses including influenza A virus and influenza B virus can be detected only by RT-PCR reaction after adding the sample to the RT-PCR reaction solution. , it is possible to inspect with sufficient sensitivity while suppressing the occurrence of non-specific reactions to a high degree.
  • the present invention is also effective in detection from samples that may contain contaminants or insoluble substances, and thus has the advantage of being able to be easily tested without prior extraction of nucleic acids. Since it is possible to simultaneously test for multiple types of respiratory infection viruses in a single test, the virus test work will be more efficient. Improving the efficiency of testing for viruses such as influenza virus can shorten the time required to provide accurate treatment to patients, and increase the number of tests for subjects who are infected but do not show symptoms. It can also greatly contribute to disease prevention.
  • FIG. 1 is a diagram showing the results of Test Example 2.
  • FIG. 1 is a diagram showing the results of Test Example 2.
  • the present invention provides a method for examining the presence or absence of target nucleic acids derived from influenza A virus and influenza B virus in a sample in one PCR reaction solution, comprising at least the following (1) to Providing a method comprising the step of (3): (1) preparing a combination of specific primer sets; (2) mixing the combination of the primer set, the sample, and the PCR reaction solution; (3) A step of performing a PCR reaction after sealing the reaction container.
  • the step (1) of the present invention by preparing and using a combination of specific primer sets, the occurrence of non-specific reactions can be highly suppressed. This is based on the discovery that influenza A and B viruses can be detected with high sensitivity even when biological samples containing contaminants such as swabs are used as they are.
  • the method of the present invention is characterized by being a multiplex RT-PCR method that detects two or more target nucleic acids.
  • a target nucleic acid can be a nucleic acid region intended to be detected by nucleic acid amplification.
  • the region intended for amplification in each genomic nucleic acid of those viruses when testing for the presence of nucleic acids from more than one virus, it may be the region intended for amplification in each genomic nucleic acid of those viruses.
  • the PCR reaction solution contains an internal control or the like, the region intended to be amplified in the internal control nucleic acid can also be the target nucleic acid.
  • the number of targets to be processed is not particularly limited as long as it is two or more. For example, it can be three, four, or five or more. Although the upper limit of the number of targets is not particularly limited, it can be set to 10 or less, for example.
  • a primer set (also referred to as a primer pair) used in the present invention includes a pair of two primers in which one primer is mutually complementary to the DNA extension product of the other primer.
  • a primer set of degenerate primers may be included when the target nucleic acid consists of subtypes.
  • Primer sets used for the detection of influenza virus include the sequences described in the "Influenza Diagnosis Manual (4th Edition)” published by the National Institute of Infectious Diseases. (Combination of primer sets of SEQ ID NOS: 39, 40, 42, 43), “Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes” (combination of primer sets of SEQ ID NOs: 31 to 34, 36, and 37), sequences described in "WHO information for the molecular detection of influenza viruses” published by the World Health Organization (WHO) (SEQ ID NOS: 45, 46,48,49 primer set combinations) are known. However, it has not been known so far to use a primer set published by each institution in combination with a primer set published by another institution.
  • WHO World Health Organization
  • primer set a and group (II) primer set combinations characterized by using any combination selected from the combination of primer sets: (I) Primer set a and primer set b (II) Primer Set f and Primer Set g (III) primer set c (IV) Primer set e and primer set g (V) Primer set d (VI) Primer set e and primer set f
  • primer sets a to g are as follows: (Primer set a): a set of nucleic acid primers consisting of the base sequences shown in SEQ ID NO: 31 and SEQ ID NO: 33, or a set of nucleic acid primers consisting of base sequences complementary to these base sequences, (primer set b): a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NO: 32 and SEQ ID NO: 34, or
  • a nucleic acid primer consisting of a nucleotide sequence represented by SEQ ID NO: 45 and a nucleic acid primer represented by SEQ ID NO: 46 corresponding to primer set d of group (V) A set of a pair of nucleic acid primers consisting of a nucleic acid primer consisting of a base sequence of the group (VI), a nucleic acid primer consisting of a base sequence represented by SEQ ID NO: 36 and a base represented by SEQ ID NO: 37 corresponding to the primer set e of group (VI) and a set of a pair of nucleic acid primers composed of nucleic acid primers consisting of sequences.
  • at least two pairs of primer sets selected from each of the above groups should be included.
  • any combination of the above primer sets can be used as long as the effect of the present invention is exhibited. is preferred, and a combination of primer set d selected from group (V) and primer set e selected from group (VI) is more preferred.
  • the concentration of the combination of the primer sets in the PCR reaction solution is not particularly limited as long as the effects of the present invention are achieved. , is preferably 0.1 ⁇ M or more and 3 ⁇ M or less. In a specific embodiment, for example, the concentration of the forward primer is 0.1 ⁇ M or more and 2 ⁇ M or less relative to the entire reaction solution, and the concentration of the reverse primer is 0.5 ⁇ M or more and 2 ⁇ M or less relative to the entire reaction solution. can.
  • a target nucleic acid derived from SARS-CoV-2 coronavirus for detecting the presence or absence of a target nucleic acid derived from SARS-CoV-2 coronavirus, together with a combination of specific primer sets for detecting influenza A virus and influenza B virus.
  • An RT-PCR reaction can be performed additionally using at least one pair of primer sets.
  • the above primer sequences detect the nucleocapsid protein (N) region of SARS-nCOV-2 according to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17. Also, the RNA-dependent RNA polymerase (RdRp) region is detected by SEQ ID NOs: 19, 20, 22, 23, 25, and 26. Also, the envelope protein (E) region is detected by SEQ ID NOS:28 and 29.
  • nucleocapsid (N) region In the detection of coronaviruses including SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame A gene such as an (ORF) region can be targeted for detection, but is not particularly limited to this.
  • a set of nucleic acid primers comprising base sequences complementary to the base sequences of the nucleic acid primers in the above combinations of primer sets can also be used.
  • any of the above primer sets can be used, but a primer set that can detect by amplifying a part of the nucleocapsid region is preferable.
  • the number of primer sets is not particularly limited, and a pair of primer sets. Alternatively, two or more pairs of primer sets may be used in combination.
  • one pair of them is a set of nucleic acid primers consisting of the base sequences shown in SEQ ID NOS: 16 and 17, or a set of these base sequences, respectively A set of nucleic acid primers comprising complementary nucleotide sequences is preferred.
  • the concentration of the combination of primer sets for detecting SARS-CoV-2 coronavirus in the PCR reaction solution is not particularly limited, but for example, the concentration of each nucleic acid primer that constitutes the primer set is It is preferably 0.1 ⁇ M or more and 3 ⁇ M or less for the entire PCR reaction solution.
  • the forward primer concentration can be 0.1 ⁇ M or more and 2 ⁇ M or less
  • the reverse primer concentration can be 0.5 ⁇ M or more and 2 ⁇ M or less, based on the total reaction mixture.
  • a combination of primer sets for detecting influenza A virus and influenza B virus as described above, and, if necessary, SARS-CoV-2 coronavirus as described above is detected.
  • PCR reaction is performed by mixing the primer set, the sample, and the PCR reaction solution.
  • the influenza virus (and, if necessary, SARS-CoV-2 coronavirus) to be tested by the present invention has RNA as the target nucleic acid, so RT-PCR reaction is performed as the PCR reaction.
  • the PCR reaction solution in the step (2) is an RT-PCR reaction solution containing a DNA polymerase having both contaminant resistance and reverse transcription activity, or an RT-PCR reaction containing a contaminant-resistant DNA polymerase and a reverse transcriptase.
  • a liquid is preferred.
  • the RT-PCR reaction may be a two-enzyme reaction system containing both reverse transcriptase and DNA polymerase, or a one-enzyme reaction system containing a thermostable DNA polymerase having reverse transcription activity. good.
  • the steps (1) to (3) are preferably carried out in the same container. That is, it is preferable not to transfer all or part of the mixture to another container during any of the steps (1) to (3). Furthermore, in step (3), it is preferable not to open and close the lid of the reaction vessel after the reaction vessel is sealed.
  • the sample to be mixed in the step (2) is a sample that may contain contaminants and insoluble substances such as saliva, nasal swabs, and pharyngeal swabs that have not undergone nucleic acid isolation treatment
  • the sample should be prepared in advance.
  • a saliva sample or the like may be used as it is, or a solid sample such as a stool sample may be directly added to the PCR reaction solution.
  • the present invention targets at least the presence or absence of influenza A virus and influenza B virus in a sample, but also other target nucleic acids (for example, viruses causing respiratory diseases such as SARS-CoV-2 coronavirus). can be inspected for the presence or absence of
  • the present invention is characterized by detecting target nucleic acids derived from two or more viruses (influenza A virus and influenza B virus) that cause respiratory infections.
  • the number of respiratory infection viruses to be tested is not particularly limited as long as it is two or more, and may be three or more.
  • the upper limit of the number of respiratory infection viruses to be tested is not particularly limited, but may be, for example, 10 or less. From the viewpoint of enabling more accurate and highly sensitive detection, 2 to 3 types of respiratory infection viruses are preferable.
  • Influenza viruses are known to cause serious respiratory illnesses, as well as complications such as bronchitis, pneumonia, otitis media, and acute encephalopathy.
  • Two types of glycoproteins called hemagglutinin (HA) and neuraminidase (NA) are present on the surface of influenza virus particles and are involved in human infection.
  • HA hemagglutinin
  • NA neuraminidase
  • viruses that can be the subject of the present invention include enveloped RNA viruses.
  • Enveloped RNA viruses include, but are not limited to, Coronaviridae viruses (eg, SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus).
  • Coronaviridae viruses eg, SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus.
  • it is the detection of coronaviruses that are difficult to diagnose from symptoms, especially coronaviruses (SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus), especially SARS-CoV-2 coronavirus, It is useful for detecting influenza A virus and human influenza B virus.
  • Non-Patent Document 2 Non-Patent Document 3
  • Non-Patent Document 4 a method for detecting SARS-CoV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases. In these techniques, detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample.
  • RNA extraction and purification steps of viral RNA are complicated and require a lot of working hours.
  • rapid one-step RT-PCR without RNA extraction and purification steps is available from biological and environmental swab samples such as pharyngeal/nasal swabs, saliva, sputum, and fecal samples containing coronaviruses, especially SARS-CoV-2.
  • Reagents and the like that can detect the
  • investigation of reagents that can detect influenza A and B viruses and SARS-CoV-2 with high sensitivity directly in one-step RT-PCR from biological samples that have not undergone such a purification process is still insufficient. I can't say.
  • the method of the present invention further comprises the step of detecting the nucleic acid amplification products amplified by each of the primer sets described above, using identifiable probes.
  • a nucleic acid probe labeled with a fluorescent compound can be used as the probe.
  • at least one labeled hybridization probe and a double-stranded DNA-binding fluorescent compound can be combined for detection using two or more fluorescent compounds.
  • Double-stranded DNA-binding fluorescent compounds include, for example, SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (registered trademark; Biotium) , LC Green (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science), and the like.
  • Hybridization probes include, for example, TaqMan probes (U.S. Pat. Nos. 5,210,015, 5,538,848, 5,487,972, 5,804 , 375), molecular beacons (U.S. Pat. No. 5,118,801), FRET hybridization probes (WO 97/46707, WO 97/46712, WO 97/46714 pamphlet), etc. Preferred are TaqMan probes.
  • any fluorescent compound known in the art can be used as the fluorescent compound that can be used for the hybridization probe.
  • fluorescent compounds include rhodamine (ROX) or derivatives thereof (e.g., 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA )), or rhodamine compounds such as salts thereof; fluorescein or derivatives thereof (e.g., FAM (carboxyfluorescein), JOE (6-carboxy-4′,5′-dichloro2′,7′-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5′-hexachloro-fluorescein-CE phosphoramidite)), VI
  • a quenching substance suitable for the fluorescent substance to be used can be used as the fluorescent compound, if necessary.
  • quenching substances corresponding to the above fluorescent substances include TAMRA (tetramethyl-rhodamine), DABCYL (4-(4-dimethylaminophenylazo)benzoic acid), BHQ1 (BHQ: Black Hole Quencher (registered trademark) )), BHQ2, BHQ3, and the like, but are not limited thereto.
  • the nucleotide sequences of the nucleic acid probes used for the detection of influenza viruses include the sequences (SEQ ID NOs: 41 and 44) described in the "Influenza Diagnosis Manual (4th Edition)” published by the National Institute of Infectious Diseases. "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes” (SEQ ID NOS: 35, 38) announced by the Centers for Disease Control and Prevention, announced by the World Health Organization (WHO)
  • the sequences described in "WHO information for the molecular detection of influenza viruses” (SEQ ID NOS: 47 and 50) can be mentioned, and can be preferably used in the present invention, but are not limited thereto.
  • the fluorescence-labeled probe for detecting influenza A virus is a nucleic acid probe consisting of a nucleotide sequence represented by any of SEQ ID NO: 35, SEQ ID NO: 41, or SEQ ID NO: 47 or a nucleotide sequence complementary thereto.
  • Preferred fluorescence-labeled probes for detecting type B influenza virus are nucleic acid probes consisting of the base sequence shown in any of SEQ ID NO: 38, SEQ ID NO: 44, or SEQ ID NO: 50 or a base sequence complementary thereto. obtain.
  • the present invention preferably further uses probes capable of detecting coronaviruses (eg, SARS-CoV-2 coronavirus).
  • coronaviruses eg, SARS-CoV-2 coronavirus.
  • the nucleotide sequences of the SARS-CoV-2 coronavirus detection probes include the sequences (SEQ ID NOS: 3 and 6) described in the "Pathogen Detection Manual 2019-nCoV” published by the National Institute of Infectious Diseases, and the American disease "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers and Probes" (SEQ ID NOS: 9, 12, 15) and “Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes" (SEQ ID NO: 18) "Research Use Only 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Primers and Probes” (SEQ ID
  • the probe sequences described above detect the nucleocapsid protein (N) region of SARS-CoV-2 according to SEQ ID NOs: 3, 6, 9, 12, 15, and 18. Also, the RNA-dependent RNA polymerase (RdRp) region is detected by SEQ ID NOs: 21, 24, and 27. Also, the envelope protein (E) region is detected by SEQ ID NO:30. Furthermore, if the targeted nucleic acid consists of subtypes, it may contain degenerate sequences. In the detection of coronaviruses such as SARS-CoV-2, genes such as the N region, E region, S region, RdRp region, and ORF region can be targeted for detection, but are not particularly limited to these. do not have.
  • nucleic acid probe capable of detecting a nucleic acid amplification product of the nucleocapsid region
  • the nucleic acid probe consists of the nucleotide sequence of SEQ ID NO: 3, 6, 9, 12, 15 or 18 or a nucleotide sequence complementary thereto. is more preferable.
  • the nucleic acid probe may be labeled with the fluorescent compound described above.
  • the fluorescently labeled nucleic acid probe may be added to the PCR reaction solution after performing the RT-PCR reaction in the step (3), or the PCR reaction solution used in the step (2) may contain the fluorescently labeled nucleic acid probe. may contain. More conveniently, it is preferable to use a PCR reaction solution containing a fluorescently labeled nucleic acid probe in the step (2).
  • concentration of such nucleic acid probes is not particularly limited, for example, it is preferably 0.01 ⁇ M or more and 1.0 ⁇ M or less, and preferably 0.015 ⁇ M or more and 0.75 ⁇ M or less, relative to the entire PCR reaction solution. More preferably, it is 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • Samples used in the present invention include, for example, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, sputum, vomit, saliva, gargle, tears, feces (excretion, rectal stool) and the like, but particularly It is not limited, and can be used for all substances derived from living organisms.
  • RNA samples there is an advantage that it is not necessary to isolate RNA from these samples using a commercially available RNA purification kit or the like and subject it to RT-PCR reaction.
  • the sample may be directly subjected to detection, or a sample in which the sample is suspended in water, physiological saline, or a buffer solution in order to reduce the influence of contaminants on the reaction and obtain more stable test results.
  • a buffer solution include, but are not limited to, Hank's buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and tricine buffer.
  • the sample may be treated with a sputazyme enzyme solution, although not particularly limited.
  • the sample used in the present invention is mixed with a solution containing a buffer solution, an acid or alkaline solution, an organic solvent, etc., in order to facilitate the extraction of RNA or DNA from robust structures such as capsids and cell walls. It may be a sample that has been
  • the acidic solution contained in the solution is not particularly limited as long as it is an acidic solution.
  • acidic solutions include aqueous formic acid, aqueous acetic acid, aqueous butyric acid, aqueous hydrochloric acid, aqueous nitric acid, aqueous sulfuric acid, aqueous citric acid, aqueous lactic acid, aqueous phosphoric acid, aqueous benzoic acid, aqueous oxalic acid, aqueous tartaric acid, and aqueous ascorbic acid. , an aqueous sulfonic acid solution, etc., and can be used singly or in combination of two or more.
  • the alkaline solution is not particularly limited as long as it is an alkaline solution.
  • alkaline solutions include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, calcium carbonate.
  • organic solvents include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphorictriamide, dimethylsulfoxide, acetone, acetonitrile, ethanol, and methanol. , 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto.
  • the sample used in the present invention may be subjected to heat treatment after being mixed with the solution.
  • the conditions for the heat treatment are not particularly limited, but may be 60° C. or higher, preferably 70° C. or higher, more preferably 80° C. or higher, and still more preferably 90° C. or higher for 1 second or longer.
  • a sample of another aspect of the present invention is a swab test sample.
  • Wiping inspections are useful for clarifying the contamination route and grasping the contamination status of the facility environment.
  • the wiping test is not particularly limited, but for example, the relevant section or equipment is wiped with a cotton swab or the like, eluted in water or a buffer solution, and concentrated by polyethylene glycol (PEG) precipitation.
  • Specific examples of swabbing test procedures include "Improvement of swabbing test method for norovirus" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html). It is not particularly limited, and includes a wide range of similar methods.
  • Examples of areas to be wiped include kitchen utensils such as cutting boards, kitchen knives, dish towels, tableware, refrigerator handles, toilets, bathroom doorknobs, washrooms, kitchens, toilets, bathroom faucets, cooks' hands and fingers, and bathrooms. , toilets, washrooms, handrails, living rooms, and other facilities.
  • kitchen utensils such as cutting boards, kitchen knives, dish towels, tableware, refrigerator handles, toilets, bathroom doorknobs, washrooms, kitchens, toilets, bathroom faucets, cooks' hands and fingers, and bathrooms.
  • toilets, washrooms, handrails, living rooms, and other facilities Moreover, although it is not a wiping test, it can also be applied to a concentrated sewage sample as an environmental test. Since these test samples contain a large amount of dirt and dust at the test site, this technique with enhanced contamination resistance in samples that can contain contaminants and insoluble substances is beneficial for these tests.
  • one feature of the method of the present invention is to use a sample that has not undergone nucleic acid isolation treatment.
  • nucleic acids are isolated from various samples using commercially available nucleic acid purification kits, or genomic nucleic acids are exposed from viral structures (e.g., cell membrane, capsid structure) by prior heat treatment or the like.
  • No untreated sample can be used. It is preferable to use these untreated samples from the viewpoint of convenience because no troublesome pretreatment is required.
  • it may be a sample that has undergone nucleic acid extraction without separation and purification to remove contaminants.
  • the sample subjected to nucleic acid extraction without separation and purification means that the nucleic acid is exposed in the sample.
  • nucleic acids can be amplified satisfactorily even in samples prepared without such isolation and purification, and it is possible to stably obtain test results for a plurality of respiratory infections.
  • Such a nucleic acid extraction treatment without separation and purification can be performed prior to the step (2).
  • Contaminants and insoluble substances that may be contained in the sample used in step (2) include feces (excretion, rectal stool), vomit, saliva, sputum, gargle, nasal swab, pharyngeal swab, and nasopharyngeal swab. , tear fluid, blood, and those derived from swab test samples, but are not limited to those derived from living organisms and can be used for environmental test samples in general, especially feces (excretion, rectal stool), saliva, sputum, gargle, tear, pharyngeal swab, nasopharyngeal swab, nasal swab.
  • the concentration of insoluble substances that can be contained varies depending on the test sample, but if it is contained in the PCR reaction solution or RT-PCR reaction solution at a turbidity of OD660, for example, 0.01 Abs / ⁇ L or more, the test sensitivity may be affected.
  • the turbidity OD660 may be 0.05 Abs/ ⁇ L or more, 0.1 Abs/ ⁇ L or more, 0.5 Abs/ ⁇ L or more, or 1 Abs/ ⁇ L or more, but is not particularly limited.
  • the upper limit of the concentration of the insoluble substance that can be contained is not particularly limited as long as the effects of the present invention are exhibited. According to the present invention, it may be possible to detect two or more target nucleic acids with two or more types of fluorescent compounds even when a high turbidity test sample is mixed with a PCR reaction solution.
  • the work of purifying nucleic acids derived from microorganisms from samples is a cause of complication and an increase in work time.
  • the work of dispensing the PCR reaction solution into a reaction container such as a PCR tube or a PCR plate is a work that can be performed hundreds or thousands of times depending on the number of samples. Continuous dispensing work into the reaction container may cause mistakes such as omission of dispensing into the reaction container and multiple dispensing. These mistakes make it impossible to carry out the inspection correctly, and cause additional work such as re-inspection, resulting in loss of time and money.
  • the present invention simplifies the work at the work site in such virus inspections and enables rapid inspections, thereby preventing further spread of infection.
  • the PCR cycle in the step (3) is 1. 2. heat treatment; A reverse transcription reaction step may be included. Moreover, before and after each step, a heat treatment step for activating the hot start enzyme may be included.
  • One heat treatment step can include disrupting the virus to expose the nucleic acid within the virus and/or activating hot start enzymes in a nucleic acid amplification reaction.
  • the temperature and time of the heat treatment step may be 60° C. or higher and 1 second or longer, preferably 70° C. for 30 seconds or longer, more preferably 80° C. for 30 seconds or longer, and particularly preferably 85° C. for 30 seconds or longer. seconds or more.
  • the temperature of the reverse transcription reaction in 2 is determined by the reverse transcription activity of the reverse transcriptase used and the Tm values of the primers and probes, and may be at least 25°C or higher. More preferably, it is 37°C or higher.
  • [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of primers to template single-stranded DNA, [3] the above using DNA polymerase Extension of the primer may be included, and [2] and [3] may be performed at the same temperature to form two steps.
  • the thermal cycler used for the RT-PCR reaction has a total elongation time of steps [2] and [3] of 15 seconds or less, more preferably 10 seconds or less. It is desirable to set up a measurement program for As used herein, the term "PCR elongation time" refers to the time set in the thermal cycler.
  • any DNA polymerase known in the art can be used as the DNA polymerase contained in the PCR reaction solution.
  • any DNA polymerase known in the art that is resistant to contaminants can be used.
  • Contaminant resistance refers to the property of a DNA polymerase having high enzymatic activity sufficient for nucleic acid amplification reaction even in the presence of PCR inhibitors.
  • the contaminant-resistant DNA polymerase is not particularly limited, but includes Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEP VENT, HawkZ05, and mutants thereof. Examples include, but are not limited to.
  • Tth SEQ ID NO: 51
  • HawkZ05 SEQ ID NO: 52
  • Tth or variants thereof DNA polymerases such as Taq, which normally do not have contamination resistance, can be used if they are mutants that have contamination resistance due to amino acid mutation.
  • the total amount of the contaminant-resistant DNA polymerase contained in the PCR reaction solution may be at least 4.2 ng/ ⁇ L or more, preferably 5.0 ng/ ⁇ L or more, and 5.8 ng/ ⁇ L. It is more preferable to be above. Among them, it is preferably 8.3 ng/ ⁇ L or more.
  • the upper limit of the total amount of the contaminant-resistant DNA polymerase is not particularly limited, but as an example, it can be 20 ng/ ⁇ L or less, and even if it is 16.7 ng/ ⁇ L or less, the effect of the present invention can be sufficiently obtained. can be done.
  • the amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be estimated from the Safety Data Sheet (SDS). When protein such as BSA is included, it is desirable to calculate by the latter method.
  • enzymatic activity of DNA polymerase is suppressed until the start of PCR reaction by using it in combination with an anti-DNA polymerase antibody or introducing a thermolabile blocking group into DNA polymerase by chemical modification. , preferably applicable to hot-start PCR.
  • the PCR reaction solution used in step (2) contains DNA polymerase and, if necessary, reverse transcriptase.
  • the origin of the reverse transcriptase contained in the PCR reaction solution is not particularly limited as long as it can convert RNA into DNA.
  • -RT EIAV-RT
  • Carboxydothermus hydrogenoformam DNA polymerase and variants thereof.
  • Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.
  • the PCR reaction solution used in the step (2) may contain a DNA polymerase that also has reverse transcriptase activity.
  • a DNA polymerase with reverse transcription activity is a DNA polymerase that has both the ability to convert RNA into cDNA and the ability to amplify DNA.
  • a DNA polymerase having reverse transcription activity preferably has heat resistance in addition to reverse transcription activity and contaminant resistance.
  • heat resistance means that the enzymatic activity does not decrease by more than half even after heat treatment at 70°C for 1 minute or more.
  • DNA polymerase derived from Thermus aquaticus (Taq)
  • DNA polymerase derived from Thermus thermophilus HB8 (Tth)
  • DNA polymerase derived from Thermus sp Z05 Z05
  • DNA polymerase derived from Thermotoga maritima have been used.
  • thermostable DNA polymerase that also has Especially preferred are DNA polymerases having reverse transcription activity selected from the group consisting of Tth, Z05 and variants thereof.
  • a DNA polymerase mutant having contaminant resistance refers to, for example, 85% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence of the wild-type DNA polymerase from which it is derived. Further preferably, it has a sequence identity of 98% or more, particularly preferably 99% or more, and has a high DNA polymerase activity even in the presence of contaminants. In the case of a DNA polymerase that also has a reverse transcription activity, it has the activity of converting RNA into cDNA and the activity of amplifying DNA even in the presence of contaminants.
  • any method known in the art can be used to calculate the identity of amino acid sequences.
  • BLAST Basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • Mutants that can be used in the present invention have one or several amino acid substitutions, deletions, insertions and/or additions (hereinafter collectively referred to as " It may be a polypeptide consisting of an amino acid sequence that has been mutated (also referred to as “mutated”), and have the same activities as wild-type DNA polymerase to convert RNA into cDNA and to amplify DNA.
  • 1 or several may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, still more preferably 1 to 5, but is not particularly limited.
  • the PCR reaction solution used in the present invention includes a buffer, an appropriate salt, a magnesium salt or a manganese salt, deoxynucleotide triphosphates, a virus to be detected, or a detection target region of a nucleic acid derived from a virus. It may contain a pair of primers that are compatible with each other and, if necessary, additives.
  • the buffer used in the present invention is not particularly limited, but includes Tris, Tricine, Bis-Tricine, Bicine and the like. It is adjusted to pH 6-9, more preferably pH 7-9, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, or the like. Moreover, the concentration of the added buffer is 10 to 200 mM, preferably 20 to 150 mM.
  • a salt solution is added to provide suitable ionic conditions for the reaction. Salt solutions include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, and the like.
  • dNTPs used in the present invention dATP, dCTP, dGTP, and dTTP are each added at 0.1 to 0.5 mM, most generally about 0.2 mM. Precautions against cross-contamination may be taken by using dUTP instead of and/or as part of dTTP. Inclusion of Uracil-N-glycosylase (UNG) is preferred when taking precautions against cross-contamination.
  • UNG Uracil-N-glycosylase
  • the PCR reaction solution preferably contains divalent cations. Containing divalent cations in this way makes it possible to obtain more stable and high contamination resistance and to perform highly sensitive detection.
  • divalent cations include, but are not limited to, magnesium ions, manganese ions, calcium ions, copper ions, iron ions, nickel ions, zinc ions, and the like.
  • magnesium ions and manganese ions are included as divalent cations.
  • magnesium ions, manganese ions, or the like are added to the PCR reaction solution, magnesium or manganese may be added, or salts thereof may be added.
  • magnesium or salts thereof include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate, etc.
  • manganese or salts thereof include manganese, manganese chloride, manganese sulfate, manganese acetate, and the like.
  • Magnesium, manganese, or a salt thereof is preferably added to the PCR reaction solution in an amount of about 1 to 10 mM. In the test method of the present invention, manganese or a salt thereof is preferably contained from the viewpoint of easily obtaining high sensitivity in a stable manner when performing single-enzyme RT-PCR.
  • the RT-PCR reaction solution preferably contains 1 mM or more manganese or a salt thereof, preferably 1.5 mM or more manganese or a salt thereof, and 2.0 mM or more manganese or a salt thereof. It is more preferable to include Moreover, when performing a two-enzyme system RT-PCR, it is preferable to contain magnesium or a salt thereof from the viewpoint of easily obtaining high sensitivity stably. In a specific embodiment, the PCR reaction solution preferably contains 1 mM or more magnesium or a salt thereof, preferably 1.5 mM or more magnesium or a salt thereof, and 2.0 mM or more magnesium or a salt thereof. is more preferred.
  • 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”
  • a polypeptide hereinafter referred to as "betaine-like quaternary ammonium”
  • BPF blocking peptide fragment
  • Glycerol glycol and surfactants.
  • the polypeptide used in the present invention is not particularly limited as long as it has a molecular weight of 5-500 kDa, but preferably 6-400 kDa.
  • molecular weights when molecular weights are indicated, they refer to values determined using SDS-PAGE, unless clearly indicated otherwise.
  • the measurement of molecular weight by SDS-PAGE can be carried out using commercially available molecular weight markers and the like using techniques and devices commonly used in the field.
  • a "molecular weight of 50 kDa” refers to a range in which a person skilled in the art normally determines that there is a band at the position of 50 kDa when the molecular weight is measured by SDS-PAGE.
  • the polypeptide used in the present invention may be a mixture of polypeptides within the above molecular weight range.
  • the polypeptide used in the present invention is not particularly limited as long as the effect of the present invention is exhibited, and refers to a protein formed by connecting a plurality of amino acids via peptide bonds.
  • the polypeptide used in the present invention may be, for example, a heat-denatured polypeptide (eg, gelatin) whose three-dimensional structure is unfolded by heat denaturation or the like, as long as it has a polypeptide structure in which amino acids are linked.
  • polypeptides that can be used in the present invention include, for example, albumin (eg, bovine serum albumin, lactalbumin, human serum albumin, egg-derived albumin), gelatin (eg, fish gelatin, porcine gelatin), sericin, Naturally derived proteins (naturally derived polypeptides) such as casein and fibroin; blocking peptide fragments (hereinafter also referred to as BPF), collagen hydrolysates, polypeptones, yeast extracts, beef extracts, etc. artificially produced by synthesis/decomposition Polypeptides and the like can be used.
  • albumin eg, bovine serum albumin, lactalbumin, human serum albumin, egg-derived albumin
  • gelatin eg, fish gelatin, porcine gelatin
  • sericin Naturally derived proteins (naturally derived polypeptides) such as casein and fibroin
  • Naturally derived proteins naturally derived polypeptides
  • BPF blocking peptide fragments
  • the polypeptide used in the present invention is preferably bovine serum albumin, gelatin, blocking peptide fragment (hereinafter BPF), and/or sericin.
  • BPF blocking peptide fragment
  • Bovine serum albumin and gelatin are more preferably used from the viewpoint that a high effect can be exhibited even in a small amount.
  • These polypeptides may be used alone or in combination of two or more.
  • these polypeptides may be prepared by means of extraction from nature, synthesis, or the like, and commercially available products can also be suitably used.
  • the amount of the polypeptide used is not particularly limited as long as the effect of the present invention is achieved.
  • the final concentration is 0.0001 to 200 mg / mL, preferably 0.01 to 150 mg / mL, more preferably 0.1 to 130 mg / mL, more preferably 0.5 to 100 mg / mL can do.
  • a preferred amount for exhibiting a better effect may vary depending on the type of polypeptide used, the degree of desired effect, and the like. For example, the following amounts can be used.
  • the final concentration in the PCR reaction solution is, for example, 0.5 mg/mL or higher, preferably 1 mg/mL or higher, more preferably 2 mg/mL or higher, and still more preferably 3 mg/mL or higher. Although the upper limit is not particularly limited, it can be, for example, 10 mg/mL or less.
  • final concentration in the PCR reaction solution is, for example, 0.1 mg/mL or higher, preferably 1 mg/mL or higher, more preferably 5 mg/mL or higher, even more preferably 7.5 mg/mL or higher, and even more preferably is greater than or equal to 15 mg/mL.
  • the upper limit is not particularly limited, it can be, for example, 50 mg/mL or less.
  • the final concentration in the PCR reaction solution is, for example, 1 mg/mL or higher, preferably 5 mg/mL or higher, more preferably 10 mg/mL or higher, even more preferably 20 mg/mL or higher, and even more preferably 50 mg/mL. that's all.
  • the upper limit is not particularly limited, it can be, for example, 100 mg/mL or less.
  • final concentration in the PCR reaction solution is, for example, 1 mg/mL or more, preferably 5 mg/mL or more, more preferably 10 mg/mL or more, still more preferably 20 mg/mL or more, and even more preferably 30 mg/mL that's all.
  • the upper limit is not particularly limited, it can be, for example, 50 mg/mL or less.
  • Surfactants contained in the PCR reaction solution include Triton X-100, Triton X-114, Tween20, Nonidet P40, Briji35, Briji58, SDS, CHAPS, CHAPSO, Emulgen 420, etc., but not particularly limited.
  • concentration of the surfactant in the PCR reaction solution is not particularly limited, it is preferably 0.0001% or more, more preferably 0.002% or more, and still more preferably 0.005% or more, and good detection is possible. becomes.
  • the upper limit is not particularly limited, it can be 0.1% or less as an example.
  • betaine-like quaternary ammonium contained in the PCR reaction solution examples include betaine (trimethylglycine) and carnitine, but are not particularly limited.
  • the betaine structure is a stable compound with both positive and negative charges in the molecule, and it is thought to exhibit surfactant-like properties and cause destabilization of the virus structure.
  • a preferred concentration of said betaine-like quaternary ammonium is 0.1M to 2M, more preferably 0.2M to 1.2M.
  • Facilitators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur or acetic acid containing compounds, dimethylsulfoxide (DMSO), glycerol, ethylene Glycol, propylene glycol, trimethylene glycol, formamide, acetamide, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), Examples include, but are not limited to, polyethylene glycol.
  • PVP polyvinylpyrrolidone
  • TMAC tetramethylammonium chloride
  • TMAH tetramethylammonium hydroxide
  • TMAA tetramethylammonium acetate
  • Examples include, but are not limited
  • ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane- A chelating agent such as N,N,N',N'-tetraacetic acid (BAPTA) may be included.
  • kits containing The kit of the present invention comprises any combination selected from the following group (I) and group (II) combinations; group (III) and group (IV) combinations; or group (V) and group (VI) combinations.
  • a primer set combination comprising: (I) Primer set a and primer set b (II) Primer Set f and Primer Set g (III) primer set c (IV) Primer set e and primer set g (V) Primer set d (VI) Primer set e and primer set f
  • the primer sets a to f are the same as those described for the method of testing for the presence or absence of target nucleic acids derived from influenza A virus and influenza B virus in a sample using one PCR reaction solution. obtain.
  • the at least two pairs of primer sets may be included in the kit as one reagent, or may be included in the kit as separate reagents and provided in a manner that they are mixed and prepared before use.
  • the present invention may also be provided in the form of a composition comprising a combination of the primer sets.
  • the kit or composition of the present invention may contain other components (for example, the type and amount of other primer sets or nucleic acid probes, the type and amount of contaminant-resistant DNA polymerase, the kit or composition
  • the type of sample used for the test using , the virus to be tested, etc. can be the same as those detailed in the test method.
  • these other components are also Either or both of the above two pairs of primer sets may be included in the kit as one reagent, or may be included in the kit as separate reagents.
  • Test example 1 Evaluation of Primer Sets for Detection of SARS-CoV-2 Coronavirus and Influenza Virus (Negative Control) (1-1) Reaction solution Detection of SARS-CoV-2 coronavirus and influenza virus type A or B in the reaction solution in one-step RT-PCR using the reaction solution and enzyme solution having the compositions shown below. reaction was performed. ⁇ Reaction solution and enzyme solution (SARS-CoV-2 Detection Kit -Multi- (Toyobo) accessories)
  • the reaction solution and enzyme solution were mixed with the primer/probe solution described in (2-2) below to prepare an RT-PCR reaction solution with a final volume of 40 ⁇ L.
  • 8 ⁇ L of sterilized water and 3 ⁇ L of pretreatment solution SARS-CoV-2 Detection Kit -Multi- (Toyobo) accessory
  • pretreatment solution SARS-CoV-2 Detection Kit -Multi- (Toyobo) accessory
  • the primer set and probe for detecting SARS-CoV-2 coronavirus are a forward primer consisting of the nucleotide sequence of SEQ ID NO: 16, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 17, and a fluorescence-labeled nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 18. used the probe.
  • the primer set and probe for detecting influenza A virus used a forward primer consisting of the nucleotide sequence of SEQ ID NO: 31, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 33, and a fluorescence-labeled nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 35.
  • a forward primer consisting of the nucleotide sequence of SEQ ID NO: 36, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 37, and a fluorescence-labeled probe consisting of the nucleotide sequence of SEQ ID NO: 38 were used as the primer set and probe for detecting influenza B virus.
  • TaqMan® corresponding to SARS-CoV-2 coronavirus (ROX channel), influenza A (CY5 channel) and B (FAM channel), internal control (NED channel).
  • ROX channel SARS-CoV-2 coronavirus
  • influenza A CY5 channel
  • B FAM channel
  • NBD channel internal control
  • the primer set and probe for detecting SARS-CoV-2 coronavirus are a forward primer consisting of the nucleotide sequence of SEQ ID NO: 16, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 17, and a fluorescence-labeled nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 18. was used.
  • the primer set and probe for detecting influenza A virus used a forward primer consisting of the nucleotide sequence of SEQ ID NO: 45, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 46, and a fluorescence-labeled nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 47.
  • the primer set and probe for detecting type B influenza virus used a forward primer consisting of the base sequence of SEQ ID NO: 36, a reverse primer consisting of the base sequence of SEQ ID NO: 37, and a fluorescence-labeled nucleic acid probe consisting of the base sequence of SEQ ID NO: 38. .
  • TaqMan® corresponding to SARS-CoV-2 coronavirus (ROX channel), influenza A (Cy5 channel) and B (FAM channel), internal control (NED channel).
  • ROX channel SARS-CoV-2 coronavirus
  • influenza A Cy5 channel
  • B FAM channel
  • NBD channel internal control
  • Results Table 1 shows the measurement results when negative control reactions were performed using each primer set.
  • Primer Set 1 was used under the present test conditions, some influenza A virus signals were detected in spite of the negative control.
  • primer set 2 was used, only the internal control was detected, confirming that the occurrence of non-specific reactions could be highly suppressed.
  • Test example 2 Multiplex detection study of SARS-CoV-2 coronavirus and influenza virus in the presence of specimen (1-1) Preparation of each virus RNA SARS-CoV-2 coronavirus (SeraCare) and influenza virus type A (Vircell), type B (Vircell) template RNA was prepared in sterilized water to 500, 50 and 25 copies/ ⁇ L, respectively.
  • reaction solution and enzyme solution were mixed with the primer/probe solution described in (2-2) below to prepare an RT-PCR reaction solution with a final volume of 40 ⁇ L.
  • RT-PCR reactions were performed.
  • the primer set and probe for detecting SARS-CoV-2 coronavirus are a forward primer consisting of the nucleotide sequence of SEQ ID NO: 16, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 17, and SEQ ID NO: 18
  • the primer set and probe for detecting influenza A virus used a forward primer consisting of the nucleotide sequence of SEQ ID NO: 45, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 46, and a fluorescence-labeled nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 47.
  • the primer set and probe for detecting type B influenza virus used a forward primer consisting of the base sequence of SEQ ID NO: 36, a reverse primer consisting of the base sequence of SEQ ID NO: 37, and a fluorescence-labeled nucleic acid probe consisting of the base sequence of SEQ ID NO: 38.
  • TaqMan® probes corresponding to SARS-CoV-2 coronavirus (ROX channel), influenza A (Cy5 channel) and B (FAM channel) were utilized for detection of each amplicon. Concentrations of probes and primers in the RT-PCR reaction solution were added as described in the same document.
  • the present invention can be suitably used in clinical examinations, examinations aimed at preventing the spread of infectious diseases, as well as molecular biological research.

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Abstract

La présente invention concerne un ensemble d'oligonucléotides permettant de détecter des acides nucléiques cibles dérivés d'une pluralité de virus, y compris les virus de la grippe de type A et de type B, avec une sensibilité élevée par PCR. La présente invention concerne les éléments suivants : une trousse pour détecter la présence d'acides nucléiques cibles dérivés du virus de la grippe de type A et du virus de la grippe de type B dans un échantillon, la trousse comprenant un ensemble d'amorces d'acide nucléique selon une combinaison particulière comprenant des amorces d'acide nucléique comprenant une séquence de bases représentée par l'une quelconque des SEQ ID NO : 31 à 34, 36 à 37, 39 à 40, 42 à 43, 45 à 46, ou 48 à 49, ou comprenant une séquence de bases complémentaire à cette dernière ; un procédé d'utilisation de la trousse ; etc.
PCT/JP2022/013261 2021-03-29 2022-03-22 Ensemble d'oligonucléotides pour détecter une pluralité de types de virus par pcr multiplex WO2022210122A1 (fr)

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