WO2022050141A1 - Procédé de test pour infection respiratoire utilisant une pcr multiplex - Google Patents

Procédé de test pour infection respiratoire utilisant une pcr multiplex Download PDF

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WO2022050141A1
WO2022050141A1 PCT/JP2021/031095 JP2021031095W WO2022050141A1 WO 2022050141 A1 WO2022050141 A1 WO 2022050141A1 JP 2021031095 W JP2021031095 W JP 2021031095W WO 2022050141 A1 WO2022050141 A1 WO 2022050141A1
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virus
dna polymerase
sample
coronavirus
pcr reaction
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PCT/JP2021/031095
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Japanese (ja)
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謙太 寺内
千恵 川井
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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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for detecting nucleic acid derived from a pathogenic microorganism of a respiratory infection such as a virus by nucleic acid amplification. More specifically, the present invention relates to detection of nucleic acid derived from a pathogenic microorganism by adding a reaction solution of a real-time reverse transcription-polymerase chain reaction without isolating and purifying the nucleic acid in advance from a sample.
  • the method of the present invention detects nucleic acids derived from two or more pathogenic microorganisms in, for example, pharyngeal and nasal swab samples, saliva and sputum samples, mouthwash, tears, blood samples, fecal samples, environmental wipe samples and the like. It is possible.
  • the present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection and the like.
  • Nucleic acid amplification method is a technology that amplifies several 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 and the like.
  • 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 elongation as one cycle. Annealing and elongation may be performed at the same temperature in two steps.
  • RT reverse transcription
  • This RT-PCR is a one-enzyme system one-step RT in which (1) RT and PCR are carried out discontinuously, two-step RT-PCR, and (2) RT and PCR are carried out continuously using a single enzyme.
  • -PCR (3) Using two types of enzymes, reverse transcriptase and DNA polymerase, RT and PCR are roughly divided into three types: two-enzyme system 1-step RT-PCR.
  • Coronavirus which is one of the pathogenic RNA viruses, is a typical example of virus testing. Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). It is known that those that cause fatal and serious respiratory diseases such as -nCOV-2 or SARS-CoV-2) occur.
  • Non-Patent Document 1 One method of accelerating the identification of pathogenic microorganisms that cause respiratory infections is to test against multiple types of pathogenic microorganisms at the same time in a single test.
  • Non-Patent Document 1 In recent years, in genetic testing for respiratory tract infections, multiplex PCR targeting nucleic acids derived from a plurality of pathogenic microorganisms has been used.
  • Non-Patent Document 2 Report that highly pathogenic North American pig genital respiratory syndrome viral RNA can be detected directly from pig serum samples by RT-PCR.
  • PCR reaction inhibitors vary greatly depending on the type of sample. For example, insoluble substances such as polysaccharides and food waste in saliva samples, and polysaccharides and blood in pharyngeal and nasal swabs are brought in as PCR reaction inhibitors.
  • norovirus detection kit G1 / G2-high-speed probe detection- has been sold as a test kit capable of detecting viral RNA by RT-PCR without omitting the RNA extraction and purification steps.
  • this test kit it is possible to simultaneously detect two types of targets, G1 type and G2 type, which are different genotypes within the same species.
  • the inactivation and nucleic acid extraction conditions of pathogenic microorganisms such as viruses and the PCR reaction conditions optimized for them (for example, reaction solution composition, temperature cycle conditions, etc.) differ greatly depending on the type. Has been done. Therefore, when targeting a plurality of types of pathogenic microorganisms, the detection sensitivity is often different for each pathogenic microorganism, and simultaneous measurement is difficult.
  • coronavirus particularly SARS-nCOV-2
  • influenza virus which have similar symptoms of respiratory diseases and are difficult to diagnose from the symptoms. It is desired to develop a method capable of rapidly and simultaneously detecting these viruses by one-step RT-PCR without RNA extraction and purification steps from pharyngeal and nasal swabs, saliva, fecal samples, wiping environment samples and the like.
  • An object of the present invention is to use a simple method while having excellent workability when a sample containing a sample (so-called crude sample) containing a contaminating substance or an insoluble substance that inhibits a nucleic acid amplification reaction or detection of a nucleic acid amplification product is used. It is to provide a method capable of detecting the presence or absence of a target nucleic acid derived from a plurality of pathogenic microorganisms with high sensitivity.
  • a sample that has not been isolated from nucleic acid and may contain a contaminating substance and / or an insoluble substance is obtained as a PCR reaction solution containing a specific DNA polymerase.
  • PCR reaction including RT-PCR reaction
  • target nucleic acids for example, target nucleic acids derived from different pathogenic microorganisms such as viruses and bacteria.
  • nucleic acids derived from a plurality of pathogenic microorganisms can be obtained by simply adding nucleic acids to a PCR reaction solution and performing a multiplex PCR reaction without performing isolation and purification work of nucleic acids from a sample that may contain contaminants in advance. We have found that it is detectable and have completed the present invention.
  • Typical inventions of the present application are as follows.
  • [Item 1] A method for inspecting a sample for the presence or absence of target nucleic acid derived from two or more pathogenic microorganisms causing respiratory tract infection with one PCR reaction solution, which comprises the following steps: (1) A step of mixing a sample that has not been isolated from nucleic acid and a PCR reaction solution containing a DNA polymerase having resistance to contaminants. (2) A step of carrying out a PCR reaction after sealing the reaction vessel; and (3) A step of detecting two or more target nucleic acids with two or more types of fluorescent compounds.
  • the PCR reaction solution in the step (1) is an RT-PCR reaction solution containing a DNA polymerase having both contamination resistance and reverse transcription activity, or a DNA polymerase having contamination resistance and a reverse transcriptase.
  • the sample is at least one selected from the group consisting of saliva sample, sputum sample, mouthwash, tear fluid, pharyngeal swab sample, nasal swab sample, fecal sample and wiping test sample, Item 1 or The method according to 2.
  • the sample is a suspension suspended in at least one selected from the group consisting of water, physiological saline, a buffer solution, and a sputazyme enzyme solution, or a centrifugal supernatant or a concentrate thereof.
  • Item 8. The method according to any one of Items 1 to 3.
  • the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.
  • the DNA polymerase having contamination resistance is a DNA polymerase having at least one contaminant resistance selected from the group consisting of Tth, HowkZ05 and variants thereof. The method according to any one of 5.
  • [Item 7] A DNA polymerase consisting of an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 39) or HawkZ05 polymerase (SEQ ID NO: 40) and having contamination resistance.
  • Item 6. The method according to Item 6, which exhibits activity.
  • 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: 39) or HawkZ05 polymerase (SEQ ID NO: 40).
  • the reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof.
  • MMRV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • Item 8 The method according to any one of Items 2 to 8.
  • Item 10 The method according to any one of Items 1 to 9, wherein at least one kind of the fluorescent compound is a double-stranded DNA-bound fluorescent compound.
  • Double-stranded DNA-bound fluorescent compounds are SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen (registered trademark; Biotium), LCGreen, Item 10.
  • Item 12. The method according to any one of Items 1 to 11, wherein at least one of the fluorescent compounds is a hybridization probe.
  • Item 13 The method according to Item 12, wherein the hybridization probe is a TaqMan probe.
  • [Item 14] The method according to any one of Items 1 to 13, wherein the two or more kinds of fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.
  • Pathogenic microorganisms that cause respiratory infections include coronavirus, influenza virus, RS virus, adenovirus, rhinovirus, parainfluenza virus, chlamydia, mycoplasma, krebsiera, tuberculosis, pertussis, and pneumonia.
  • Item 6. The method according to any one of Items 1 to 14, which is at least two selected from the group consisting of bulbous bacteria. Item 6.
  • the coronavirus family virus is at least one selected from the group consisting of SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and SARS-CoV-2 coronavirus.
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • SARS-CoV-2 coronavirus SARS-CoV-2 coronavirus.
  • the method according to any one of Items 1 to 18, wherein the two or more pathogenic microorganisms causing respiratory infections include at least an orthomyxoviridae virus.
  • Item 20 The method according to Item 19, wherein the Orthomyxoviridae virus is at least one selected from the group consisting of influenza A virus, influenza B virus, and influenza C virus.
  • the Orthomyxoviridae virus is at least one selected from the group consisting of influenza A virus, influenza B virus, and influenza C virus.
  • Item 2. The method according to any one of Items 1 to 20, which comprises.
  • the PCR reaction solution is an RT-PCR reaction solution containing a DNA polymerase having both contamination resistance and reverse transcription activity, or a DNA polymerase having contamination resistance and a reverse transcriptase.
  • the kit or composition described.
  • Item 24 The kit or composition according to Item 22 or 23, wherein the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.
  • the DNA polymerase having contamination resistance is a DNA polymerase having at least one contaminant resistance selected from the group consisting of Tth, HowkZ05 and variants thereof.
  • a DNA polymerase consisting of an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 39) or HawkZ05 polymerase (SEQ ID NO: 40) and having contamination resistance.
  • Item 25 The kit or composition according to Item 25, which exhibits activity.
  • 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: 39) or HawkZ05 polymerase (SEQ ID NO: 40).
  • Item 5 The kit or composition according to Item 25 or 26, which exhibits DNA polymerase activity having contamination resistance.
  • the reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof.
  • MMRV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • Double-stranded DNA-bound fluorescent compounds are SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen®, LCGreen, Item 29.
  • the kit or composition according to Item 31, wherein the hybridization probe is a TaqMan probe.
  • the two or more kinds of fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.
  • a sample that has not been isolated of microbial nucleic acid and may contain contaminants and insoluble substances is added to a PCR reaction solution (including an RT-PCR reaction solution), and then a PCR reaction (RT-PCR reaction) is performed.
  • a PCR reaction solution including an RT-PCR reaction solution
  • RT-PCR reaction PCR reaction
  • One aspect of the present invention is, for example, an examination of pathogenic microorganisms (including viruses, bacteria, fungi, etc.) in a sample, in which nucleic acids (DNA, RNA) of pathogenic microorganisms are isolated and purified from the sample.
  • a PCR reaction solution including RT-PCR reaction solution
  • a DNA polymerase having contamination resistance is added to a sample containing a contaminant to two or more pathogenic microorganisms that cause respiratory infections. It is a method for examining the presence of pathogenic microorganisms, which comprises detecting different target nucleic acids derived from two or more kinds of fluorescent compounds.
  • a method for examining the presence or absence of a target nucleic acid derived from a pathogenic microorganism causing two or more respiratory infections in a sample of the present invention with one PCR reaction solution is characterized by comprising at least the following steps. .. (1) A step of mixing a sample that has not been isolated from nucleic acid and a PCR reaction solution containing a DNA polymerase having resistance to contaminants. (2) A step of carrying out a PCR reaction after sealing the reaction vessel; and (3) A step of detecting two or more target nucleic acids with two or more types of fluorescent compounds.
  • the target nucleic acid derived from the pathogenic microorganism to be tested is RNA
  • an RT-PCR reaction is performed as a PCR reaction.
  • the PCR reaction solution of the step (1) is an RT-PCR reaction solution containing a DNA polymerase having both contamination resistance and reverse transcription activity, or an RT-PCR containing a DNA polymerase having contamination resistance and a reverse transcriptase. It can be a reaction solution.
  • the RT-PCR reaction is carried out in the step (2), even if it is a two-enzyme reaction system containing both reverse transcriptase and DNA polymerase, it is a one-enzyme reaction system containing a heat-resistant DNA polymerase having reverse transcription activity. You may. It is preferable that the steps (1) to (3) are performed in the same container. That is, it is preferable not to transfer all or a part of the mixed solution to another container during any of the steps (1) to (3). Furthermore, in step (2), preferably in both steps (2) and (3), it is preferable not to open or close the reaction vessel lid after sealing the reaction vessel.
  • the sample that can contain the contaminating substance and the insoluble substance used in the step (1) may be a suspension suspended in water or a buffer solution in advance, and if it is a saliva sample or the like, it may be used as it is.
  • a solid sample such as a fecal sample may be added directly to the PCR reaction solution.
  • the test target in the present invention may be, for example, a nucleic acid derived from a pathogenic microorganism, but is not particularly limited.
  • the pathogenic microorganism refers to any microorganism that can infect living organisms such as humans and other mammals and cause infectious diseases, and is not limited to prokaryotes such as bacteria or fungi and eukaryotes. It is a concept that includes viruses and the like.
  • the invention is characterized by the detection of target nucleic acids derived from two or more pathogenic microorganisms that cause respiratory infections.
  • the number of pathogen microorganisms for respiratory infections to be inspected is not particularly limited as long as it is 2 or more, and may be 3 or more, 4 or more, or 5 or more.
  • the upper limit of the number of pathogen microorganisms for respiratory infections 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, it is preferably 2 to 4 types of respiratory tract infection pathogenic microorganisms, and more preferably 2 to 3 types of respiratory tract infection pathogenic microorganisms. More preferably, there are two types of respiratory infection pathogenic microorganisms.
  • pathogenic microorganisms include Escherichia coli (eg, enterohemorrhagic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EIEC), toxinogenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EAEC), enterohemorrhagic Escherichia coli (EHEC)).
  • Escherichia coli eg, enterohemorrhagic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EIEC), toxinogenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EAEC), enterohemorrhagic Escherichia coli (EHEC)
  • EEC enterohemorrhagic Escherichia coli
  • EIEC enterohemorrhagic Es
  • Campylobacter Campylobacter, Welsh, Salmonella, Listeria, Botulinum, Seleus, Pseudomonas (multi-drug resistant Escherichia coli), Clostridium, Regionella, Streptococcus, yellow Escherichia coli (eg, methicillin-resistant Staphylococcus aureus), Acinetobacta spp. Examples thereof include S. coli, Pest, Riquetia, Chlamydia, Escherichia coli, Toxoplasma, Mycoplasma, and Klebsiella, but are not particularly limited.
  • the present invention can be used to test for any pathogenic microorganisms that can cause respiratory infections, but in particular respiratory infections such as tuberculosis, pneumonia, pertussis, chlamydia, mycoplasma, and Klebsiella, which are difficult to diagnose from symptoms.
  • respiratory infections such as tuberculosis, pneumonia, pertussis, chlamydia, mycoplasma, and Klebsiella
  • the simple inspection method of the present invention which has excellent workability, is particularly useful.
  • the pathogenic microorganism may be a virus.
  • the virus to be the subject of the present invention may be a DNA virus or an RNA virus.
  • DNA viruses include herpesviridae virus (eg, simple herpesvirus, cytomegalovirus); adenoviridae virus (eg, human adenovirus); hepadonaviridae virus (eg, hepatitis B virus); papovaviridae virus. (Eg, papillary virus, polyomavirus); parvoviridae virus (eg, adeno-associated virus, human parvovirus); asfaviridae virus (eg, African pig fever virus), etc., but are particularly limited. It's not a thing.
  • herpesviridae virus eg, simple herpesvirus, cytomegalovirus
  • adenoviridae virus eg, human adenovirus
  • hepadonaviridae virus eg, hepatitis B
  • the present invention can be used to test for any pathogenic microorganisms that can cause respiratory infections, but in particular, the detection of pathogens that cause respiratory infections, such as adenoviridae viruses, which are difficult to diagnose from symptoms.
  • the inspection method of the present invention which has excellent workability and is simple, is particularly useful.
  • the RNA virus may be an RNA virus having no envelope derived from the lipid double membrane or an RNA virus having an envelope.
  • non-enveloped RNA viruses include astroviridae virus (eg, astrovirus); caliciviridae virus (eg, sapovirus, norovirus); picornaviridae virus (eg, hepatitis A virus, echovirus, etc.). Enterovirus, coxsackie virus, poliovirus, rhinovirus); hepevirus family virus (eg, hepatitis E virus); leovirus family virus (eg, rotavirus), etc., but not limited to, preferably.
  • Calisivirus and Leovirus viruses are useful for the detection of Calisivirus and Leovirus viruses, more preferably for the detection of norovirus, sapovirus, and rotavirus, and particularly useful for the detection of norovirus.
  • Most non-enveloped viruses can infect the gastrointestinal tract due to fecal-oral infection, etc., and RNA is retained in a rigid capsid structure that is resistant to inactivation by gastric acid and the surface-active action of bile acids. Therefore, conventionally, since the genetic test for norovirus includes a virus RNA extraction and purification step, the simple test method of the present invention having excellent workability is particularly useful.
  • the present invention can be used to test for any non-enveloped RNA virus that can cause respiratory infections, but in particular, causes respiratory infections such as picornaviruses, especially rhinoviruses, which are difficult to diagnose from symptoms.
  • the inspection method of the present invention which has excellent workability and is simple, is particularly useful for detecting the pathogenic bacterium.
  • Envelope RNA viruses include flaviviridae viruses (eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus); Togaviridae virus (eg, ruin virus, chikungnia virus); coronaviridae virus (eg, coronavirus family virus).
  • flaviviridae viruses eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus
  • Togaviridae virus eg, ruin virus, chikungnia virus
  • coronaviridae virus eg, coronavirus family virus.
  • SARS Coronavirus SARS Coronavirus, MERS Coronavirus, SARS-CoV-2 Coronavirus
  • Orthomixoviridae virus eg, influenza virus
  • Rabdoviridae virus eg, mad dog disease virus
  • Bunyaviridae virus eg, Crimea congo
  • Fever virus Hunter virus
  • Paramyxoviridae virus eg, parainfluenza virus, measles virus, human RS virus
  • Phylloviridae virus eg, Ebola virus
  • the present invention can be used to test for any enveloped RNA virus that can cause respiratory infections, but is particularly difficult to diagnose from symptoms: coronaviridae virus, orthomyxoviridae virus, paramyxoviridae virus, among others.
  • the present invention is preferably carried out to test for two or more viruses selected from these.
  • it is the detection of coronavirus family virus and orthomyxoviridae virus, and more preferably coronavirus (SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus) and influenza virus (influenza virus type A, B type, C type), and is particularly useful for the detection of SARS-nCOV-2 coronavirus and at least one influenza virus selected from type A influenza virus and type B human influenza virus.
  • Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). -It is known that those that cause fatal and serious respiratory diseases such as CoV-2) occur. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronavirus is important in clinical diagnosis, food hygiene inspection, environmental inspection, and the like.
  • nucleic acid amplification technology capable of detecting coronavirus with high sensitivity is widely used.
  • Several techniques have been developed for detecting coronavirus by nucleic acid amplification method (for example, Non-Patent Document 2, Non-Patent Document 3, Patent Document 2).
  • RNA extraction and purification steps from biological samples such as pharyngeal / nasal swabs containing coronavirus, especially SARS-CoV-2, saliva, sputum, and fecal samples, and wiping environment samples. It is desired to develop a method that can detect the virus.
  • Influenza virus like coronavirus, is the causative virus that causes respiratory infections. In addition to often causing serious respiratory illness, it is known to cause bronchitis, pneumonia, otitis media, acute encephalopathy, etc. as complications. There are three main types of influenza, type A, type B, and type C, and it is type A and type B that show the epidemic spread every year. There are two types of glycoproteins called hemagglutinin (HA) and neuraminidase (NA) on the surface of influenza virus particles, which are involved in human infection. There are 15 different subtypes of HA and 9 different subtypes of NA, and viruses with various combinations of these are widely distributed. Due to the regular appearance of different subtype combinations of the two glycoproteins described above, influenza A is a worldwide pandemic every few to decades.
  • HA hemagglutinin
  • NA neuraminidase
  • Influenza virus and SARS-CoV-2 coronavirus are also known to cause serious respiratory illness and have very similar symptoms. Since the treatment method differs for each virus, it is necessary to identify the causative virus at an early stage. This is because the present invention can easily and highly sensitively detect target nucleic acids derived from two or more pathogenic microorganisms that cause respiratory infections, while being a method having excellent workability and operability. Influenza virus and coronavirus (SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus, and other coronaviruses that tend to become severe), especially SARS-CoV-2 virus and influenza virus can be tested at the same time. It is beneficial.
  • Examples of the sample used in the present invention include pharyngeal swab, nasal swab, sputum, vomitus, saliva, mouthwash, tears, feces (excretion and rectal stool), but are not particularly limited. It can be used for all things derived from living organisms. In particular, it is useful for detection from pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, feces, cultured cells, and culture supernatants, especially saliva samples.
  • the sample may be subjected to direct detection, or the sample may be suspended in water, saline or buffer in order to reduce the influence of impurities on the reaction and obtain more stable test results. There may be.
  • 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 sample may be a sample treated with a sputazyme enzyme solution, although not particularly limited.
  • the sample in order to facilitate the extraction of RNA or DNA from a robust structure such as a capsid or a cell wall, the sample is mixed with a buffer solution, an acid or alkaline solution, a solution containing an organic solvent, or the like. It's okay to have it.
  • the acidic solution contained in the solution is not particularly limited as long as it is an acidic solution.
  • the acidic solution examples include formic acid aqueous solution, acetic acid aqueous solution, butyric acid aqueous solution, hydrochloric acid aqueous solution, nitrate aqueous solution, sulfuric acid aqueous solution, citric acid aqueous solution, lactic acid aqueous solution, phosphoric acid aqueous solution, benzoic acid aqueous solution, oxalic acid aqueous solution, tartaric acid aqueous solution, and ascorbic acid aqueous solution. , Sulphonic acid aqueous solution and the like, and can be used alone or in combination of two or more.
  • the alkaline solution is not particularly limited as long as it is an alkaline solution.
  • alkaline solution examples 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, and calcium carbonate.
  • the organic solvent include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethylsulfoxide, acetone, acetonitrile, ethanol and methanol. , 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto.
  • the sample added in the step (1) may be one that has been subjected to heat treatment after being mixed with the solution.
  • the conditions of the heat treatment are not particularly limited, but may be those treated at 60 ° C. or higher, preferably 70 ° C. or higher, more preferably 80 ° C. or higher, still more preferably 90 ° C. or higher for 1 second or longer.
  • the sample in the present invention is a wipe inspection sample.
  • Wiping inspection is useful for clarifying the pollution route and grasping the pollution status 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 into water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitate or the like. ..
  • PEG polyethylene glycol
  • wipes include cutting boards, kitchen knives, kitchen utensils, tableware and other cooking utensils, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilet, washbasin, handrail, living room 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. Since these test samples contain a large amount of dirt and dust at the test site, this method with enhanced contamination resistance in samples that may contain contaminants and insoluble substances is useful for these tests.
  • the method of the invention is characterized by the use of a sample that has not been treated with nucleic acid isolation.
  • the present invention may isolate nucleic acids from various samples with a commercially available nucleic acid purification kit, or may undergo prior heat treatment to expose genomic nucleic acids from the structure of pathogenic microorganisms (eg, cell membranes, capsid structures). Untreated samples can be used. It is preferable to use these untreated samples from the viewpoint of simplicity because it eliminates the need for time-consuming pretreatment. Further, the sample may be a sample obtained by nucleic acid extraction without separation and purification to remove contaminants.
  • the sample from which nucleic acid extraction is performed without separation and purification means that the nucleic acid is exposed in the sample, for example, the cell membrane, capsid, envelope, etc. are destroyed in the sample, and these are used. Extracting and exposing the encapsulated nucleic acid (however, do not remove the cell membrane, capsid, envelope fragment, etc. remaining after destruction).
  • nucleic acid can be satisfactorily amplified even in a sample prepared without the trouble of isolation and purification, and it becomes possible to stably obtain test results for a plurality of respiratory tract infections.
  • the nucleic acid extraction process that does not involve such separation and purification can be performed prior to the step (1).
  • Contaminants and insoluble substances that can be contained in the sample to be subjected to PCR or RT-PCR in the step (1) include feces (excretion stool, rectal stool), vomitus, saliva, sputum, mouthwash, nasal swab, and pharynx.
  • feces excretion stool, rectal stool
  • vomitus saliva, sputum, mouthwash, nasal swab
  • pharynx Contaminants and insoluble substances that can be contained in the sample to be subjected to PCR or RT-PCR in the step (1)
  • feces excretion stool, rectal stool
  • vomitus saliva
  • saliva sputum
  • mouthwash swab
  • nasal swab Contaminants and insoluble substances that can be contained in the sample to be subjected to PCR or RT-PCR in the step (1)
  • the concentration of insoluble substances that can be contained varies depending on the test sample, but if the turbidity OD660 is contained in the PCR reaction solution or the RT-PCR reaction solution, for example, 0.01 Abs / ⁇ L or more, the test sensitivity may be affected. However, it is not limited to this.
  • the turbidity OD660 may be 0.05 Abs / ⁇ L or more, 0.1 Abs / ⁇ L or more, 0.5 Abs / ⁇ L or more, and 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, but may be, for example, 5 Abs / ⁇ L or less and 3Abs / ⁇ L or less, but is not limited. According to the present invention, even if such a high turbidity test sample is mixed with a PCR reaction solution, it may be possible to detect two or more target nucleic acids with two or more kinds of fluorescent compounds.
  • the work of purifying nucleic acid derived from a microorganism from a sample causes the work to be complicated and the work time to be extended.
  • the work of dispensing the reaction solution of PCR into a reaction vessel such as a PCR tube or a PCR plate can be hundreds or thousands of times depending on the number of samples.
  • mistakes such as omission of dispensing into the reaction vessel and multiple dispensing may occur. These mistakes make it impossible to carry out the inspection correctly, and further work is required due to re-inspection, etc., resulting in time and financial loss.
  • in the inspection of such pathogenic microorganisms by simplifying the work at the work site and enabling the inspection to be carried out promptly, it is possible to prevent further spread of infection.
  • the PCR cycle in step (2) is 1. Heat treatment and 2. It may include a step of reverse transcription reaction. In addition, a heat treatment step for activating the hot start enzyme may be included before and after each step.
  • the heat treatment step 1 may include disrupting the virus to expose the nucleic acid in the virus and / or activating the hot start enzyme in the 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. and 30 seconds or longer, more preferably 80 ° C. and 30 seconds or longer, and particularly preferably 85 ° C. and 30 seconds. More than a second.
  • the temperature of the reverse transcription reaction of 2 is determined by the reverse transcriptase activity of the reverse transcriptase used and the Tm value of the primer and the probe, and may be at least 25 ° C. or higher. More preferably, it is 37 ° C. or higher.
  • PCR of 3 [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of primers to template single-stranded DNA, and [3] the above-mentioned using DNA polymerase. It suffices to include three steps of primer extension, and [2] and [3] may be carried out at the same temperature to form two steps.
  • the thermal cycler used for the RT-PCR reaction has a total extension time of the steps [2] and [3] of 15 seconds or less, more preferably 10 seconds or less. It is desirable to set the measurement program of.
  • the "PCR extension time" refers to the set temperature in the thermal cycler.
  • DNA polymerase contained in the PCR reaction solution any DNA polymerase known in the art can be used as long as it has contamination resistance.
  • Contaminant resistance refers to the property of having high enzymatic activity of DNA polymerase sufficient for nucleic acid amplification reaction even in the presence of PCR inhibitor.
  • DNA polymerases having resistance to contaminants include, but are not limited to, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, HowkZ05 and variants thereof. However, it is not particularly limited. Preferably, Tth (SEQ ID NO: 39) and HawkZ05 (SEQ ID NO: 40) or variants thereof are used.
  • the use of Tth or a variant thereof is particularly preferred.
  • a DNA polymerase such as Taq, which normally does not have contamination resistance, can be used if it is a mutant having contamination resistance due to an amino acid mutation.
  • the total amount of the DNA polymerase having resistance to contaminants 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. The above is more preferable. Above all, it is preferably 8.3 ng / ⁇ L or more.
  • the upper limit of the total amount of the DNA polymerase having contamination resistance 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 a protein such as BSA is contained, it is desirable to calculate by the latter method.
  • the enzymatic activity of DNA polymerase until the start of PCR reaction is suppressed by introducing it into DNA polymerase of heat unstable block group in combination with anti-DNA polymerase antibody or by chemical modification. , It is preferable that it can be applied to hot start PCR.
  • the PCR solution used in step (1) contains DNA polymerase and optionally reverse transcriptase.
  • the origin of the reverse transcriptase contained in the PCR reaction solution is not particularly limited as long as RNA can be converted into DNA, but is MMLV (Mloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV-RT, RAV2. -RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and variants thereof are exemplified. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.
  • the PCR solution used in the step (1) may contain a DNA polymerase having both reverse transcriptase activity.
  • 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.
  • a DNA polymerase having reverse transcription activity is preferably thermostable in addition to reverse transcription activity and contamination resistance.
  • the heat resistance means that the enzyme activity does not decrease by more than half even if the heat treatment is carried out at 70 ° C. for 1 minute or more.
  • the origin is not particularly limited, and examples thereof include Taq, Tth, Bst, Bca, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and variants thereof.
  • DNA polymerase derived from Thermus aquaticus (Taq), DNA polymerase derived from Thermus thermophilus HB8 (Tth), DNA polymerase derived from Thermus sp Z05 (Z05), and DNA polymerase derived from Thermotoga maritima. (Tma), DNA polymerase (Bca) derived from Bacillus caldotenax, DNA polymerase (Bst) derived from Bacillus theatermophilus, etc., even if these variants do not lose their reverse transcription activity and heat resistant DNA polymerase activity. good.
  • a variant of DNA polymerase (KOD) derived from Thermococcus kodakaraensis is known to have reverse transcription activity (for example, RTX: reverse transcription xenopolymerase), and the present invention has such reverse transcriptase activity.
  • a heat-resistant DNA polymerase having both can also be used.
  • DNA polymerases having reverse transcription activity selected from the group consisting of Tth, Z05 and variants thereof.
  • the variant of DNA polymerase having contamination resistance is, for example, 85% or more, preferably 90% or more, more preferably 95% or more with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived. Further preferably, it has 98% or more, particularly preferably 99% or more of sequence identity, and has high DNA polymerase activity even in the presence of contaminants.
  • it is a DNA polymerase that also has reverse transcription activity, it means an activity that has an activity of converting RNA into cDNA and an activity of amplifying DNA even in the presence of a contaminating substance.
  • any means known in the art can be used as a method for calculating the identity of the amino acid sequence.
  • 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.
  • 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 PCR reaction solution used in the present invention includes a buffer, a suitable salt, a magnesium salt or a manganese salt, a deoxynucleotide triphosphate, a detection target region of a nucleic acid derived from a virus to be detected or a pathogenic microorganism.
  • the corresponding primer pair and, if necessary, an additive may be contained.
  • 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 As the dNTP used 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. When taking preventive measures against cross-contamination, it is preferable to include Uracil-N-gycosylase (UNG).
  • UNG Uracil-N-gycosylase
  • the PCR reaction solution contains divalent cations.
  • divalent cations include magnesium ion, manganese ion, calcium ion, copper ion, iron ion, nickel ion, and zinc ion. It is preferable to include magnesium ion and manganese ion as the divalent cation.
  • magnesium ions, manganese ions, or the like when added to the PCR reaction solution, magnesium, manganese, or the like 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. It is preferable that such magnesium, manganese, or a salt thereof is added to the PCR reaction solution in an amount of about 1 to 10 mM. When performing monoenzyme-based RT-PCR in the test method of the present invention, it is preferable to contain manganese or a salt thereof from the viewpoint that stable and high sensitivity can be easily obtained.
  • the 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. It is more preferable to include it. Further, when performing bienzyme-based RT-PCR or PCR without reverse transcription reaction, it is preferable to contain magnesium or a salt thereof from the viewpoint that stable and high sensitivity can be easily obtained. In certain embodiments, the PCR reaction solution preferably contains 1 mM or more of magnesium or a salt thereof, preferably 1.5 mM or more of magnesium or a salt thereof, and preferably contains 2.0 mM or more of magnesium or a salt thereof. Is more preferable.
  • a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in 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
  • the polypeptide used in the present invention is not particularly limited as long as it has a molecular weight of 5 to 500 kDa, but is preferably 6 to 400 kDa.
  • a molecular weight when indicating a molecular weight, it means a value determined by using SDS-PAGE unless it is clear that it has another meaning.
  • the measurement of the molecular weight by SDS-PAGE can be performed by using a method and an apparatus common in the art and using a commercially available molecular weight marker or the like.
  • “molecular weight 50 kDa” means that when the molecular weight is measured by SDS-PAGE, those skilled in the art usually determine that there is a band at the position of 50 kDa.
  • 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 by peptide bonds.
  • the polypeptide used in the present invention is, for example, a heat-denatured polypeptide (for example, gelatin) whose three-dimensional structure is solved by heat denaturation or the like as long as it has a polypeptide structure in which amino acids are linked. May be.
  • the polypeptides that can be used in the present invention include, for example, albumin (eg, bovine serum albumin, lactoalbumin, human serum albumin, egg-derived albumin), gelatin (eg, fish gelatin, pig gelatin), sericin, and the like.
  • Naturally-derived proteins such as casein and fibroin (naturally-derived polypeptides); artificially produced by synthesis / degradation of Blocking peptide fragment (hereinafter also referred to as BPF), collagen hydrolysate, polypeptone, yeast extract, beef extract, etc.
  • BPF Blocking peptide fragment
  • Polypeptides and the like can be used.
  • the polypeptides used in the present invention are preferably bovine serum albumin, gelatin, Blocking peptide fragment (hereinafter referred to as BPF), and / or sericin. From the viewpoint that a high effect can be exhibited even in a small amount, it is more preferable to use bovine serum albumin and gelatin (particularly fish gelatin).
  • BPF Blocking peptide fragment
  • sericin from the viewpoint that a high effect can be exhibited even in a small amount, it is more preferable to use bovine serum albumin and gelatin (particularly fish gelatin).
  • These polypeptides may be used alone or in combination of two or more
  • the amount of the polypeptide used is not particularly limited as long as the effect of the present invention is exhibited, but for example, in a mixed solution of a sample containing the insoluble substance and a one-enzyme system 1-step RT-PCR reaction solution.
  • 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, and further preferably 0.5 to 100 mg / mL. can do.
  • the preferable amount for exerting a more excellent effect may vary depending on the type of polypeptide used, the degree of desired effect, and the like, and for example, the following amounts can be exemplified.
  • the final concentration in the PCR reaction solution is, for example, 0.5 mg / mL or more, preferably 1 mg / mL or more, more preferably 2 mg / mL or more, still more preferably 3 mg / mL or more.
  • the upper limit is not particularly limited, but can be, for example, 10 mg / mL or less.
  • gelatin When gelatin is used: The final concentration in the PCR reaction solution is, for example, 0.1 mg / mL or more, preferably 1 mg / mL or more, more preferably 5 mg / mL or more, still more preferably 7.5 mg / mL or more, still more preferably. Is 15 mg / mL or more.
  • the upper limit is not particularly limited, but can be, for example, 50 mg / mL or less.
  • the 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, still more preferably 50 mg / mL. that's all.
  • the upper limit is not particularly limited, but can be, for example, 100 mg / mL or less.
  • the 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, still more preferably 30 mg / mL. that's all.
  • the upper limit is not particularly limited, but can be, for example, 50 mg / mL or less.
  • Examples of the surfactant contained in the PCR reaction solution include Triton X-100 (Triton X-100), Triton X-114 (Triton X-114), Tween 20 (Tween 20), Nonidet P40, Briji35, Briji58, SDS, CHAPS, and the like. CHASPO, Emulgen 420 and the like can be mentioned, but the present invention is not particularly limited.
  • the concentration of the surfactant in the 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 possible. Will be.
  • 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 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 is stable in the molecule, and exhibits properties like a surfactant, and is thought to cause destabilization of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase.
  • the preferred betaine-like quaternary ammonium concentration is 0.1 M to 2 M, more preferably 0.2 M to 1.2 M.
  • 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, dimethylsulfoxide (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 include, but are not limited to, polyethylene glycol.
  • EGTA Ethyleneglycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid
  • BAPTA a chelating agent
  • the method of the invention is characterized by a multiplex PCR that detects two or more target nucleic acids.
  • the "target nucleic acid” may 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 pathogenic microorganisms can also be used as the target nucleic acid.
  • the number of target targets is not particularly limited, but may be two or more, for example, three, four, five or more.
  • the upper limit of the number of targets is not particularly limited, but may be, for example, 10 or less.
  • Primer pairs used in the present invention include two pairs of primers in which one primer is complementary to the DNA extension product of the other primer. Although not particularly limited, in the present invention, it is preferable that two or more pairs of the above primers are contained. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate primers.
  • SEQ ID NOs: 7, 8, 10, 11, 13, 14 and "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and Probes" (SEQ ID NOs: 16 and 17). It can be preferably used in the present invention, but the present invention is not limited to this.
  • the nucleocapsid protein (N) region of SARS-nCOV-2 is detected by SEQ ID NOs: 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17.
  • nucleocapsid (N) region In the detection of coronaviruses such as SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame. Genes such as (ORF) region can be detected, but the detection is not limited thereto.
  • concentration of the primer to be used it is preferable that 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 or less with respect to the entire RT-PCR reaction solution. .. 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.
  • influenza virus which is one of the enveloped RNA viruses
  • an example of a primer pair is the "Influenza Diagnosis Manual (4th Edition)” published by the National Institute of Infectious Diseases. (SEQ ID NOs: 27, 28, 30, 31), "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR Primers and” announced by the American Center for Disease Control and Prevention. "Probes" (SEQ ID NOs: 19, 20, 21, 22, 24, 25), the sequence described in "WHO information for the influenza virus” published by the World Health Organization (WHO) (SEQ ID NOs: 33, 34).
  • WHO World Health Organization
  • influenza A is detected by SEQ ID NOs: 27 and 28, 19 and 21, 20 and 22, 33 and 34
  • influenza B is detected by SEQ ID NOs: 30 and 31, 24 and 25, 36 and 37. Detects, but is not limited to this.
  • concentration of the primer to be used it is preferable that 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 or less with respect to the entire RT-PCR reaction solution. .. 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.
  • One feature of the present invention is to detect two or more target nucleic acids by using two or more kinds of fluorescent compounds. Therefore, in the present invention, probes labeled with two or more kinds of fluorescent compounds can be used. In yet another embodiment, there is a detection method in which at least one type of labeled hybridization probe and a double-stranded DNA-bound fluorescent compound are combined to utilize two or more types of fluorescent compounds. By using two or more kinds of fluorescent compounds in this way, the analysis of the amplified product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis labor is reduced. Furthermore, it is not necessary to open the reaction vessel, and the risk of contamination can be reduced. It is also possible to identify each target nucleic acid separately by labeling each hybridization probe with a different fluorescent dye, corresponding to the type and subtype of virus and microorganism.
  • 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) and the like.
  • any fluorescent compound known in the art can be used, and for example, it can be selected according to the qPCR device to be used.
  • Specific examples of the fluorescent compound include, for example, rhodamine (ROX) or a derivative thereof (eg, 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA).
  • rhodamine-based compounds such as salts thereof; fluorosane or derivatives thereof (eg, FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro 2', 7'-dimethoxyfluoresane),.
  • FAM carboxyfluorescein
  • JOE 6-carboxy-4', 5'-dichloro 2', 7'-dimethoxyfluoresane
  • FITC fluorescein isothiocyanate
  • TET tetrachlorofluorescein
  • HEX 5'-hexachloro-fluoresane-CE phosphoroamidite
  • VIC® BODICY® series
  • rhodamine or derivatives thereof examples thereof include 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA)), Cy® dyes (eg, Cy3, Cy5), derivatives thereof, and non-rhodamine compounds such as salts thereof.
  • the fluorescent compound a quenching substance suitable for the fluorescent substance to be used can be used, if necessary.
  • quenching substance corresponding to the above-mentioned fluorescent substance examples 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.
  • coronavirus which is one of the enveloped RNA viruses
  • the base sequence of the hybridization probe for the detection is announced by the National Institute of Infectious Diseases. Sequences (SEQ ID NOs: 3 and 6) described in the "Pathogen Detection Manual 2019-nCoV” and "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers and” announced by the Centers for Disease Control and Prevention.
  • Probes SEQ ID NOs: 9 and 12 and 15
  • Research Use Only CDC Influenza SARS-CoV-2 Flu SC2
  • Multiplex Assay Real-Time RT-PCR Primers and Probes SEQ ID NO: 18
  • the probe sequence described above detects the N region of SARS-nCOV-2.
  • the target nucleic acid if it consists of subtypes, it may contain degenerate sequences.
  • genes such as N region, E region, S region, RdRp region, ORF region can be targeted for detection, but it is not particularly limited to this. do not have.
  • 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 even more preferably 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • the base sequence of the hybridization probe for the detection is "influenza diagnosis” published by the National Institute of Infectious Diseases. Sequence (SEQ ID NOs: 29, 32) described in “Manual (4th Edition)", “Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Real-Time RT-PCR” announced by the American Center for Disease Control and Prevention. The sequences (SEQ ID NOs: 35, 38) described in “Primers and Probes" (SEQ ID NOs: 23, 26) and "WHO information for the molecular detection of influenza viruses” published by the World Health Organization (WHO) are listed.
  • influenza A type is detected by SEQ ID NOs: 29 and 23, 35
  • influenza B type is detected by SEQ ID NOs: 32, 26, 38, but the present invention is not particularly limited thereto.
  • concentration of the fluorescently labeled probe used is preferably 0.01 ⁇ M or more and 1.0 ⁇ M or less with respect to the entire RT-PCR reaction solution. More preferably, it is 0.013 ⁇ M or more and 0.75 ⁇ M or less, and even more preferably 0.02 ⁇ M or more and 0.5 ⁇ M or less.
  • test kit or composition for detecting nucleic acid derived from a virus or pathogenic microorganism in a sample, which comprises a PCR reaction solution containing a DNA polymerase having contamination resistance. It is a test kit or composition for performing a test for pathogenic microorganisms by a multiplex PCR method using two or more kinds of fluorescent compounds in a sample that may contain a contaminating substance without isolating the nucleic acid.
  • the type and amount of the contaminant-resistant DNA polymerase used in this embodiment the type and amount of primer or probe, the type of sample used for the test, the pathogenic microorganism to be tested, and the like are detailed in the above-mentioned test method. It can be similar to that described.
  • Test example 1 Turbidity measurement of saliva and pharyngeal swab
  • 1-1) Collection of saliva SARS-CoV-2 coronavirus and influenza virus negative saliva specimens were suspended in sterile water at 50 w / w%.
  • 1-2 Collection of pharyngeal swab The pharynx was wiped with a special cotton swab, and the sample suspended in 1 mL of the sample transport medium (VTM) was used as the pharyngeal swab sample and suspended in sterile water to 50 w / w% for turbidity measurement. ..
  • VTM sample transport medium
  • OD660 Measurement of turbidity
  • Test example 2 Examination of multiplex detection of SARS-CoV-2 coronavirus and influenza virus in the presence of specimens (1-1) Preparation of each viral RNA Template RNAs of SARS-CoV-2 coronavirus (Twist Bioscience) and influenza virus type A (Vircell) and type B (Vircell) were prepared in sterile water so as to be 500 copies / ⁇ L each. (1-2) Collection of saliva suspension A 500 ⁇ L saliva sample confirmed to be negative for SARS-CoV-2 coronavirus and influenza virus was collected.
  • Influenza virus type A is the primer and probe set (SEQ ID NOs: 27, 28, 29) described in the "Influenza Diagnosis Manual (4th Edition)" published by the National Institute of Infectious Diseases
  • influenza virus type B is , World Health (WHO) published "WHO information for the molecular detection of influenza viruses", primers and probe sets (SEQ ID NOs: 36, 37, 38) were used.
  • TaqMan (R) probes corresponding to SARS-CoV-2 coronavirus N1 (Cy5 channel) and influenza A and B (FAM channels) were used to detect each amplification product. As the concentration of the probe and the primer in the RT-PCR reaction solution, the concentration described in the same document was added.
  • Test example 3 Multiplex detection study of SARS-CoV-2 coronavirus and influenza virus using various DNA polymerases (2-enzyme system 1-step RT-PCR reaction)
  • (1-1) Preparation of each viral RNA Template RNAs of SARS-CoV-2 coronavirus (Twist Bioscience) and influenza virus type A (Vircell) and type B (Vircell) were prepared in sterile water so as to be 500 copies / ⁇ L each.
  • influenza virus type A the primers and probe sets (SEQ ID NOs: 27, 28, 29) described in the "Influenza Diagnosis Manual (4th Edition)" published by the National Institute of Infectious Diseases, and influenza virus type B are , World Health (WHO) published "WHO information for the molecular detection of influenza viruses", primers and probe sets (SEQ ID NOs: 36, 37, 38) were used.
  • TaqMan (R) probes corresponding to SARS-CoV-2 coronavirus N1 (Cy5 channel) and influenza A and B (FAM channels) were used to detect each amplification product.
  • concentration of the probe and the primer in the RT-PCR reaction solution the concentration described in the same document was added.
  • DNA polymerase used and its variants The DNA polymerases and variants thereof shown below were used in the reactions at the concentrations listed below.
  • the notation for variants follows the one-letter abbreviation notation for amino acids.
  • the numbers are included in the name of the enzyme, and the amino acid before the change is shown on the left and the amino acid after the change is shown on the right.
  • Tth mutant (M749K) means that M (methionine) at position 749 of Tth DNA polymerase is mutated to K (lysine).
  • Enzyme used ⁇ Enzyme 1: Taq DNA polymerase (wild type) (Toyobo) ⁇ Enzyme 2: Tth DNA polymerase (wild type) (Toyobo) -Enzyme 3: Tth mutant (Q509R) -Enzyme 4: Tth mutant (Q509K) Enzyme 5: Tth mutant (E628K) Enzyme 6: Tth mutant (M749K) Enzyme 7: Tth mutant (F751Y) Enzyme 8: Tth mutant (D549G) Enzyme 9: HawkZ05 DNA polymerase (Roche) (4) PCR reaction The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-Multi- (Toyobo).
  • Test example 4 Simultaneous detection of 3 types of SARS-CoV-2 coronavirus, influenza virus A and influenza B multiplex in the presence of specimens
  • (1-1) Collection of saliva suspension A 500 ⁇ L saliva sample confirmed to be negative for SARS-CoV-2 coronavirus and influenza virus (types A and B) was collected.
  • (1-2) Collection of pharyngeal swab After wiping the pharynx with a special cotton swab, the sample suspended in 1 mL of the sample transport medium (VTM) was used as a pharyngeal swab sample.
  • VTM sample transport medium
  • pharyngeal swab sample those confirmed to be negative for SARS-CoV-2 coronavirus and influenza virus (types A and B) were used.
  • (1-3) Mixing of virus solution and saliva or pharyngeal swab AccuPlex SARS-CoV-2, Flu A / Band RSV Reference Material Kit (Saracare) was used as the virus solution.
  • 1 ⁇ L of virus solution 100 copies / ⁇ L each) was mixed with saliva, pharyngeal swab or 8 ⁇ L of sterile water (control) to obtain 9 ⁇ L of sample-containing virus solution.
  • sample-containing virus solution 9 ⁇ L of the sample-containing pretreatment liquid and 3 ⁇ L of the pretreatment liquid (SARS-CoV-2 Detection Kit-Multi- (Toyobo) attachment) were mixed and heat-treated at 95 ° C. for 5 minutes to prepare the pretreated sample liquid. ..
  • the pretreatment method performed here does not involve the nucleic acid isolation treatment.
  • Influenza viruses A and B use the primers and probe sets (SEQ ID NOs: 19-26) described in the CDC-issued "Research Use Only CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay Primers and Probes". did. TaqMan (R) probes corresponding to SARS-CoV-2 coronavirus N1, N2 (Cy5 channel), influenza A (ROX channel), and influenza B (HEX channel) were used to detect each amplified product. .. As the concentration of the probe and the primer in the RT-PCR reaction solution, the concentration described in the same document was added. (3) PCR reaction The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-Multi- (Toyobo).
  • Test example 4 Simultaneous detection of 4 types of SARS-CoV-2 coronavirus, influenza virus A, influenza B, and RS virus multiplex in the presence of specimens
  • (1-1) Collection of saliva suspension A 500 ⁇ L saliva sample confirmed to be negative for SARS-CoV-2 coronavirus, influenza virus (types A and B), and RS virus was collected.
  • (1-2) Collection of pharyngeal swab After wiping the pharynx with a special cotton swab, the sample suspended in 1 mL of the sample transport medium (VTM) was used as a pharyngeal swab sample.
  • VTM sample transport medium
  • SARS-CoV-2 coronavirus SARS-CoV-2 coronavirus, influenza virus (types A and B), and RS virus confirmed to be negative were used.
  • 1-3 Mixing of virus solution, saliva and pharyngeal swab AccuPlex SARS-CoV-2, FluA / Band RSV Reference Material Kit (Saracare) was used as the virus solution. 1 ⁇ L of virus solution (100 copies / ⁇ L each) was mixed with saliva, pharyngeal swab or 8 ⁇ L of sterile water (control) to obtain 9 ⁇ L of sample-containing virus solution.
  • sample-containing virus solution 9 ⁇ L of the sample-containing pretreatment liquid and 3 ⁇ L of the pretreatment liquid (SARS-CoV-2 Detection Kit-Multi- (Toyobo) attachment) were mixed and heat-treated at 95 ° C. for 5 minutes to prepare the pretreated sample liquid. ..
  • the pretreatment method performed here does not involve the nucleic acid isolation treatment.
  • TaqMan corresponding to SARS-CoV-2 coronavirus N1 (Cy5 channel), influenza A (ROX channel), influenza B (HEX channel), and RS virus (FAM channel)
  • a probe was used.
  • concentrations of probes and primers in the RT-PCR reaction solution were the concentrations described in the same document.
  • RS virus the final concentration of each primer was 0.3 ⁇ M and the final concentration of the probe was 0.2 ⁇ M.
  • (3) PCR reaction The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-Multi- (Toyobo).
  • the present invention is suitably used in molecular biology research, clinical tests, food hygiene control, tests for the purpose of preventing the spread of infectious diseases, and the like.

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Abstract

Un but de la présente invention est de fournir un procédé de test utilisant une PCR multiplex améliorée, le procédé étant excellent quant à sa maniabilité et capable de détecter, de manière simple et avec une sensibilité élevée, des acides nucléiques cibles dérivés d'une pluralité de micro-organismes pathogènes pouvant causer des infections respiratoires. La présente invention concerne un procédé d'utilisation d'une solution de réaction de PCR pour tester la présence ou l'absence d'acides nucléiques cibles dérivés d'au moins deux micro-organismes pathogènes causant des infections respiratoires dans des échantillons, le procédé comprenant les étapes suivantes : (1) mélange d'un échantillon dont les acides nucléiques n'ont pas été isolés, et d'une solution de réaction PCR comprenant une ADN polymérase résistante aux impuretés ; (2) réalisation d'une réaction PCR après fermeture du récipient de réaction ; et (3) détection de deux ou plusieurs acides nucléiques cibles à l'aide de deux ou plusieurs composés fluorescents.
PCT/JP2021/031095 2020-09-04 2021-08-25 Procédé de test pour infection respiratoire utilisant une pcr multiplex WO2022050141A1 (fr)

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US20210285061A1 (en) * 2020-03-09 2021-09-16 Roche Molecular Systems, Inc. Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2), influenza a and influenza b
WO2022210122A1 (fr) * 2021-03-29 2022-10-06 東洋紡株式会社 Ensemble d'oligonucléotides pour détecter une pluralité de types de virus par pcr multiplex
WO2023196870A1 (fr) * 2022-04-05 2023-10-12 Laboratory Corporation Of America Holdings Procédés, compositions et systèmes de détection de virus respiratoire
WO2024009873A1 (fr) * 2022-07-08 2024-01-11 東洋紡株式会社 Polymérase d'acide nucléique ayant une activité de transcription inverse

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JP2017131164A (ja) * 2016-01-28 2017-08-03 東洋紡株式会社 改良されたウイルス検出方法
JP2018000138A (ja) * 2016-07-06 2018-01-11 東洋紡株式会社 改良されたウイルス検出方法
WO2018198682A1 (fr) * 2017-04-26 2018-11-01 東洋紡株式会社 Procédé d'analyse de virus et kit d'analyse de virus
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
JP2020108358A (ja) * 2019-01-07 2020-07-16 株式会社リコー 標的対象を高感度に検出するための方法
JP2020115777A (ja) * 2019-01-23 2020-08-06 東洋紡株式会社 改良された核酸検出方法

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JP2017131164A (ja) * 2016-01-28 2017-08-03 東洋紡株式会社 改良されたウイルス検出方法
JP2018000138A (ja) * 2016-07-06 2018-01-11 東洋紡株式会社 改良されたウイルス検出方法
WO2018198682A1 (fr) * 2017-04-26 2018-11-01 東洋紡株式会社 Procédé d'analyse de virus et kit d'analyse de virus
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
JP2020108358A (ja) * 2019-01-07 2020-07-16 株式会社リコー 標的対象を高感度に検出するための方法
JP2020115777A (ja) * 2019-01-23 2020-08-06 東洋紡株式会社 改良された核酸検出方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210285061A1 (en) * 2020-03-09 2021-09-16 Roche Molecular Systems, Inc. Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2), influenza a and influenza b
WO2022210122A1 (fr) * 2021-03-29 2022-10-06 東洋紡株式会社 Ensemble d'oligonucléotides pour détecter une pluralité de types de virus par pcr multiplex
WO2023196870A1 (fr) * 2022-04-05 2023-10-12 Laboratory Corporation Of America Holdings Procédés, compositions et systèmes de détection de virus respiratoire
WO2024009873A1 (fr) * 2022-07-08 2024-01-11 東洋紡株式会社 Polymérase d'acide nucléique ayant une activité de transcription inverse

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