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WO2019212023A1 - Procédé de traitement de virus à arn - Google Patents

Procédé de traitement de virus à arn Download PDF

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Publication number
WO2019212023A1
WO2019212023A1 PCT/JP2019/017490 JP2019017490W WO2019212023A1 WO 2019212023 A1 WO2019212023 A1 WO 2019212023A1 JP 2019017490 W JP2019017490 W JP 2019017490W WO 2019212023 A1 WO2019212023 A1 WO 2019212023A1
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Prior art keywords
reaction solution
reverse transcriptase
nucleic acid
sample
reverse transcription
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English (en)
Japanese (ja)
Inventor
上森　隆司
裕之 松本
美和 秋友
憲介 齋藤
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Takara Bio Inc
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Takara Bio Inc
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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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12Q1/6851—Quantitative amplification
    • 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]

Definitions

  • the present invention relates to an RNA virus processing method and a nucleic acid detection method using the processing solution.
  • the present invention can be used for food hygiene inspection, environmental inspection, clinical diagnosis and the like.
  • Samples to be tested using the nucleic acid amplification method vary depending on the purpose.
  • specimens in which the causative bacterium can exist that is, urine, sputum, stool, blood, nasal fluid, vaginal secretion, and the like.
  • food sanitation inspection food or feces and urine collected from food handlers
  • environmental sanitation inspection environmental water such as soil, river water, rain water, sea water, and wipes from manufacturing equipment.
  • a rapid inspection method is required to prevent the spread of damage. It becomes a thing.
  • nucleic acid contains a large amount of substances other than nucleic acids.
  • separation and purification of nucleic acids in specimens is usually required in order to efficiently perform nucleic acid amplification reactions using enzymes.
  • There are various methods for separating and purifying the nucleic acid but it basically comprises a step of separating the nucleic acid and other substances into a solid phase and a liquid phase and separating them into solid and liquid.
  • nucleic acid is separated into solid phases by ethanol precipitation and separated by centrifugation
  • a target nucleic acid is hybridized to nucleic acids on a solid phase (for example, beads) and separated by centrifugation or magnetized substance.
  • Patent Document 1 discloses a method of performing a nucleic acid amplification reaction by directly adding a heat-treated sample by adding an alkali-containing solution to a stool suspension supernatant.
  • Patent Document 2 discloses a method in which a nucleic acid amplification reaction is carried out by directly adding a heat-treated sample by pectinase treatment to a stool suspension supernatant.
  • RT-PCR reverse transcription PCR
  • RT-PCR reverse transcription PCR
  • heat treatment is performed with a solution that does not contain reverse transcriptase, and then cDNA synthesis is performed using the treatment solution.
  • Patent Document 2 describes the necessity of heat treatment, although the risk of hydrolysis of RNA by heat treatment is described.
  • heat treatment is performed for nucleic acid extraction and reaction inhibitor deactivation, but usually a reverse transcriptase is added to the sample solution after the heat treatment.
  • the operations such as opening and closing of the reaction solution and addition of reagents are very complicated, and there is a high risk that RNA contained in the aerosol will be scattered by opening and closing the container to cause contamination between samples.
  • Patent Document 3 As a nucleic acid amplification method for detecting RNA in a sample directly in a reverse transcription PCR (RT-PCR) reaction solution, for example, in Patent Document 3, eukaryotic cells are heated in an RT-PCR buffer solution. Discloses a method for lysing cells and amplifying nucleic acids.
  • cDNA is synthesized and amplified using a small amount of cultured cells that are considered to be able to elute RNA relatively easily.
  • Patent Document 3 describes a virus having a capsid and a thick peptidoglycan. There is no description of RNA protected by a strong outer layer structure such as a prokaryotic organism having.
  • Patent Document 4 is characterized in that the sample is added to a one-step RT-PCR reaction solution containing reverse transcriptase and thermostable DNA polymerase without purifying viral RNA from the sample or heat-treating the sample.
  • a method for detecting RNA viruses in a sample is disclosed.
  • heat treatment for elution of RNA from virus is not performed, but in the examples, an additive such as a quaternary ammonium salt is included in the RT-PCR reaction solution, and the additive is RNA It is thought to be involved in the elution of.
  • An object of the present invention is to prepare a heat-resistant reverse transcriptase reaction solution and enable nucleic acid synthesis without going through a process of extracting RNA from a sample that may contain an RNA virus.
  • RNA virus in a sample a sample that has not undergone RNA purification or prior heat treatment is mixed with a thermostable reverse transcriptase.
  • a reverse transcription reaction solution By preparing a reverse transcription reaction solution and performing RNA elution and cDNA synthesis continuously in the reverse transcription reaction, the trouble of opening and closing the container and the RNA contained in the aerosol scatter and contamination between samples. has led to the development of a nucleic acid synthesis method that eliminates the risk of oxidization.
  • the present inventors directly add a sample to an RT-PCR reaction solution containing a thermostable reverse transcriptase having a DNA-dependent DNA synthesis activity, and sequentially perform RNA elution, cDNA synthesis, and nucleic acid amplification reaction.
  • the present inventors have found that viral RNA can be detected more efficiently and have completed the present invention.
  • a method for treating an RNA virus comprising the following steps; (A) preparing a sample that may contain an RNA virus; (B) a step of preparing a reverse transcription reaction solution containing the sample of step (a) and a thermostable reverse transcriptase, (C) A step of heating the reaction solution obtained in the step (b). [2] The method according to [1], wherein the reverse transcription reaction solution further contains a reagent necessary for the PCR reaction. [3] The processing method according to [1] or [2], wherein the reverse transcription reaction solution contains a surfactant.
  • thermostable reverse transcriptase is a reverse transcriptase exhibiting reverse transcription activity at 40 ° C. or higher.
  • thermostable reverse transcriptase is an enzyme having DNA-dependent DNA synthesis activity.
  • thermostable reverse transcriptase is an enzyme having DNA-dependent DNA synthesis activity.
  • RNA virus is feces, fecal suspension, or a supernatant thereof.
  • stool suspension supernatant is a supernatant prepared by suspending stool in a buffer solution, sterilized water, or physiological saline.
  • RNA virus is a caliciviridae virus.
  • a method for synthesizing cDNA comprising the following steps; (A) preparing a sample that may contain an RNA virus; (B) a step of preparing a reverse transcription reaction solution containing the sample of step (a) and a thermostable reverse transcriptase, (C) a step of heating the reaction solution obtained in step (b), (D) A step of further reacting the reaction solution obtained in the step (c) under conditions where the thermostable reverse transcriptase exhibits reverse transcriptase activity.
  • a nucleic acid amplification method comprising the following steps; (A) preparing a sample that may contain an RNA virus; (B) a step of preparing a reverse transcription reaction solution containing the sample of step (a) and a thermostable reverse transcriptase, (C) a step of heating the reaction solution obtained in step (b), (D) a step of further reacting the reaction solution obtained in the step (c) under conditions where the thermostable reverse transcriptase exhibits reverse transcriptase activity; (E) A step of further reacting the reaction solution obtained in step (d) under nucleic acid amplification conditions.
  • the nucleic acid amplification method according to [14], wherein the nucleic acid amplification in the step (e) is PCR.
  • cDNA can be synthesized from RNA of RNA virus in a sample simply by adding the sample to the reverse transcription reaction solution without providing a step of isolating and purifying viral RNA from the sample.
  • the time and cost for separating and purifying the nucleic acid prior to the nucleic acid amplification reaction can be reduced.
  • the risk that may occur when nucleic acid is separated and purified that is, the risk of sample loss and cross-contamination can be reduced.
  • the effect is remarkable in a test in which a large number of specimens using a biological material represented by feces as a sample are processed.
  • RNA virus processing method of the present invention The sample to which the processing method of the present invention can be applied is not particularly limited as long as it is a sample that may contain an RNA virus.
  • a sample collected from a living body For example, a tissue, a tissue piece, blood, etc.
  • a biologically discharged sample and a sample derived from a sample collected from the environment.
  • the biologically discharged specimen includes stool, urine, sputum, nasal discharge, nasal discharge, saliva, saliva, and the like, and these can be directly applied to the present invention as a sample.
  • these biologically discharged specimens are suspended in water (sterilized water, etc.), physiological saline, buffer solution, organic solvent, liquid medium, or gel medium (for example, Caribbean medium), and prepared from the above suspension.
  • a sample that has been subjected to some kind of treatment such as cleansing can also be applied to the method of the present invention as a sample.
  • the living body include humans, pets, domestic animals, wild animals, insects, and all other animals.
  • Feces composed of enteric bacteria, intestinal epithelial cells, food-derived substances, and the like are representative of the specimen of the present invention.
  • the biologically discharged sample may be collected from the environment.
  • the environment includes all kinds of places where an RNA virus that is desired to be detected may exist, but is not limited to the natural environment.
  • the surface of any object that may contain an RNA virus such as a facility itself such as a manufacturing facility or a cooking facility, water in the facility, drainage from the facility, or equipment of the facility is exemplified.
  • wiping operations or other known methods are used for collecting specimens from these environments.
  • the collected material is suspended in an appropriate solution such as water (sterilized water, etc.), physiological saline, buffer solution, etc., and the supernatant and the precipitate recovered from the suspension by centrifugation or the like. Applies to the method of the present invention.
  • the RNA virus to be detected by the detection method of the present invention may be any, for example, a non-envelope RNA virus.
  • non-enveloped RNA viruses include viruses belonging to the Caliciviridae family (Norovirus (NoV), Sapovirus (SV), feline Calicivirus (FCV) etc.) and viruses belonging to the Reoviridae family (Rotavirus (Rota)), Examples include viruses belonging to the Picornaviridae family (echovirus (E), enterovirus (EV)), and the like.
  • NoV Caliciviridae family
  • SV Sapovirus
  • FCV feline Calicivirus
  • FCV Reoviridae family
  • Examples include viruses belonging to the Picornaviridae family (echovirus (E), enterovirus (EV)), and the like.
  • the feline calicivirus (FCV) listed above is an alternative virus that is widely used for evaluation of disinfectants and detergents in place of human norovirus (HuNoV) that cannot be
  • One embodiment of the present invention includes a step of preparing a reverse transcription reaction solution by mixing a sample that may contain an RNA virus and a thermostable reverse transcriptase.
  • the reverse transcription reaction solution refers to a solution containing a thermostable reverse transcriptase and other elements necessary for the reverse transcription reaction.
  • thermostable reverse transcriptase used in the method of the present invention is not particularly limited to the present invention, but is an enzyme that exhibits reverse transcription activity at 40 ° C or higher, preferably an enzyme that exhibits reverse transcription activity at 50 ° C or higher. More preferably, it refers to an enzyme that exhibits reverse transcription activity at 60 ° C. or higher, and more preferably an enzyme that exhibits reverse transcription activity at 65 ° C. or higher.
  • heat-resistant mutants of virus-derived reverse transcriptases such as MMLV, HIV, RAV2, and EIAV are exemplified.
  • thermostable reverse transcriptase suitable for the present invention include reverse transcriptase derived from thermophilic bacteria and super thermostable bacteria, and DNA-dependent DNA synthesis having reverse transcription activity.
  • An active DNA polymerase can be preferably used.
  • thermus thermophilus Thermus thermophilus
  • thermus aquaticus T. aquaticus
  • bacillus stearothermophilus Bacillus stearothermophilus
  • B. caldtenax B.
  • trt gene product of Geobacillus stearothermophilus Appl. Env. Microbiol. 70, p7140-7147 (2004)] or a variant thereof.
  • Elements necessary for the reverse transcription reaction include primers for cDNA synthesis having a sequence complementary to the RNA to be detected, salts, deoxyribonucleotides, and buffer components. These elements are added to a sample together with a thermostable reverse transcriptase and mixed to prepare a reverse transcription reaction solution. MgCl 2 , KCl, or the like is used as the above-mentioned salts, but other salts may be added as appropriate or may be changed to other salts.
  • the buffer component refers to a compound or mixture having an action of reducing fluctuations in the hydrogen ion concentration (pH) of the reaction solution.
  • a mixed solution of a weak acid and its salt or a weak base and its salt has a strong buffering action, and is therefore widely used for the purpose of maintaining an appropriate pH.
  • Various reaction buffers known in the field of biochemistry can be used in the present invention, and it is appropriate that they are set within a normal range in which reverse transcription reaction or nucleic acid amplification reaction is carried out.
  • the reverse transcription reaction solution may further contain a surfactant, and a more favorable result can be obtained by adding a surfactant.
  • the surfactant is not particularly limited, and nonionic, anionic, cationic or amphoteric surfactants may be used alone or in combination.
  • the surfactant is not particularly limited, but is Triton (registered trademark) X-100 (Polyoxyethylene (10) octylphenyl ether), Tween (registered trademark) 20 (Polyoxyethylene Sorbitan (registered trademark))
  • Nonionic surfactants such as 40 (Octylphenyl-polyethylene glycol), anionic surfactants such as poly (ethyleneglycyl) 4-nonylphenyl 3-sulfopropyl ether (PNSE), and the like.
  • Surfactants and amphoteric interfaces such as cocamidopropyl betaine Examples include sex agents, among which Triton (registered trademark) X-100, Tween (registered trademark) 20, Nonidet (registered trademark) P-40, and poly (ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether are preferred examples. Is done. Since the surfactant degrades the cell membrane or softens the cell wall and capsid protein structure, it is considered that this treatment method can be suitably used for RNA elution of cells and bacteria. Furthermore, a known substance added to a reverse transcription reaction solution or a nucleic acid amplification reaction solution for the purpose of improving reactivity may be included. Examples of the substance added to the reaction solution include proteins such as bovine serum albumin (BSA), betaine, and polyanionic polymer substances, but are not limited thereto.
  • BSA bovine serum albumin
  • the reverse transcription reaction solution may further contain a reagent necessary for target nucleic acid amplification.
  • a reagent necessary for target nucleic acid amplification include, but are not limited to, nucleic acid amplification primer pairs, heat-resistant DNA polymerase, deoxyribonucleotides (dNTP), and the like.
  • One of the primer pairs for nucleic acid amplification may be common with the primer for cDNA synthesis.
  • the thermostable DNA polymerase may be a DNA polymerase having thermostable reverse transcriptase activity. It goes without saying that the composition of the reverse transcription reaction solution in this embodiment is adjusted so that each of the RNA virus treatment, reverse transcription reaction, and target nucleic acid amplification is appropriately performed.
  • RNA virus treatment method of the present invention a sample that may contain an RNA virus as described above is reacted with a reverse transcription reaction solution containing a thermostable reverse transcriptase at a high temperature.
  • the holding temperature in the reaction is 65 ° C. or more and less than 100 ° C., preferably 70 ° C. or more and less than 100 ° C., next preferred is 75 ° C. to 99 ° C., more preferably 80 ° C. to 99 ° C., more preferably It is in the range of 85 ° C to 99 ° C, particularly preferably 90 ° C to 99 ° C.
  • a suitable holding time is 30 minutes or less, preferably 15 minutes or less, more preferably 5 minutes or less, still more preferably 3 minutes or less, and particularly preferably 0 second to 1 minute.
  • holding time 0 second means stopping heating at the same time it reaches the intended temperature.
  • thermostable reverse transcriptase can be suitably used for elution of RNA from eukaryotic cells and bacteria.
  • it can be used more favorably for RNA elution of eukaryotic cells having no cell wall.
  • the DNA that is complementary to the RNA contained in the RNA virus in the sample is left as it is in the reaction solution subjected to the RNA virus treatment method of (1) above.
  • the reaction temperature in the reverse transcription reaction is not particularly limited as long as the thermostable reverse transcriptase is active. In consideration of destroying the secondary structure of RNA, it is 35 ° C. or higher and lower than 100 ° C., preferably 40 ° C. to 90 ° C., more preferably 45 ° C. to 80 ° C., and further preferably 50 ° C. to 70 ° C. It is.
  • the optimum temperature of 60 ° C. can be suitably used.
  • the reverse transcription reaction may be performed using the time for raising the temperature of the reaction solution from 70 ° C. to 89 ° C.
  • the reaction temperature is kept as high as possible within the temperature range in which the primer for cDNA synthesis can be annealed, the time required for the cDNA synthesis step can be further shortened.
  • the time for the reverse transcription reaction can be appropriately set in consideration of the chain length of the synthesized cDNA and the like.
  • Target nucleic acid amplification method of the present invention In the method of the present invention, a cDNA amplification reaction can be combined with the reverse transcription reaction of (2) above.
  • the nucleic acid amplification method is not particularly limited as long as it is a known method.
  • Polymerase Chain Reaction PCR
  • Strand Displacement Amplification SDA
  • Multiple Displacement Amplification MDA
  • Rolling-Circulation Amplification MDA
  • Rolling-Circle Amplification Examples include Amplification (LAMP), Smart Amplification Process (SmartAmp), Helicalase-Dependent Amplification (HDA), and Ligase Chain Reaction (LCR).
  • RNA elution from RNA virus, reverse transcription reaction and amplification reaction of target nucleic acid are continuously performed in the same reaction solution in the same container.
  • RT-PCR of steps can be performed.
  • the high-temperature reverse transcription reaction and the nucleic acid amplification are performed in the same container that remains closed. The reaction is carried out continuously.
  • a solution containing a thermostable reverse transcriptase and a thermostable DNA polymerase is used as this RT-PCR reaction solution.
  • thermostable DNA polymerase examples include various polymerases that can be used for PCR, such as pol I-type polymerase, ⁇ -type polymerase, and mixtures thereof.
  • a thermostable DNA polymerase having thermostable reverse transcriptase activity can be used for the RT-PCR reaction solution.
  • the enzyme having both reverse transcriptase activity and DNA-dependent DNA synthesis activity is preferably a pol I type thermostable DNA polymerase, for example, Thermus thermophilus, T. aquaticus, Bacillus stearothermophila. Examples thereof include DNA polymerases such as Bacillus stearothermophilus, B. caldotenax, and T. species Z05, and mutants thereof.
  • the RT-PCR reaction solution used in the target nucleic acid amplification method of the present invention may further include a primer pair for amplifying the target region of cDNA.
  • one of the primers used for amplification of the target nucleic acid may be shared with the reverse transcription reaction primer in the reverse transcription reaction of the previous step.
  • the RT-PCR reaction solution may contain a known substance added to a reverse transcription reaction solution or a nucleic acid amplification reaction solution for the purpose of improving reactivity.
  • the substance added to the reaction solution include proteins such as bovine serum albumin (BSA) described in (1) above, various surfactants, betaine, acidic polymers, and the like, but are not limited thereto. is not.
  • BSA bovine serum albumin
  • dNTP, dATP, dGTP, dCTP, and dTTP are usually used, but dUTP is further used as a reaction solution for the purpose of preventing contamination of DNA amplification products that are used as templates for other nucleic acid amplification reactions.
  • dTTP may be replaced with dUTP.
  • dUTP is incorporated, and uracil-N-glycosylase (UNG) having the activity of degrading uracil-containing DNA is allowed to act before the next nucleic acid amplification reaction.
  • UNG uracil-N-glycosylase
  • Nucleic acid amplification products can be degraded. In this way, it is possible to prevent contamination caused by the amplification product obtained in the previously performed reaction being mixed in the subsequent reaction.
  • the obtained amplified DNA product can be further detected and identified by electrophoresis, melting curve method, and various probe methods (Q probe, scorpion probe, hybrid probe, etc.).
  • a real-time detection method is particularly preferable from the viewpoint of preventing contamination of amplification products.
  • a TaqMan (registered trademark) probe and a Cycle (TM) probe may be used as long as the device has a temperature raising / lowering function and a fluorescence measurement function.
  • Probes having fluorescent labels and quenching labels such as SYBR (registered trademark) Green I, SYTO-60, SYTO-62, POPO-3, TOTO-3, BOBO-3, TO-PRO-3, YO-PRO- 1.
  • the amplified DNA product can be detected and identified during the progress of the reaction.
  • an intercalating dye such as SYTOX OrangeSYBR (registered trademark) or TB Green (registered trademark)
  • the amplified DNA product can be detected and identified during the progress of the reaction.
  • Kit of the present invention provides a kit used in the above-described method for amplifying a target nucleic acid of the present invention.
  • the kit of the present invention prepares a reaction solution for successively performing heating of a reverse transcription reaction solution containing a sample, cDNA synthesis by a thermostable reverse transcriptase, and amplification of a nucleic acid derived from the obtained cDNA.
  • Ingredients necessary for this purpose are contained as components.
  • a probe or an intercalator for real-time detection may be included for detecting the target nucleic acid.
  • the kit of the present invention includes, for example, a buffer component, a thermostable reverse transcriptase, and deoxyribonucleotides such as dNTP and dUTP. Further, a surfactant, salts, a primer for reverse transcription reaction and the like may be included. As another embodiment, for example, a kit containing a premix reaction solution containing a one-step buffer component, a thermostable reverse transcriptase, a thermostable DNA polymerase, a surfactant, salts, and deoxyribonucleotides such as dNTP and dUTP is exemplified. .
  • a reverse transcription reaction primer and at least one target nucleic acid amplification primer pair are contained, and an RT-PCR reaction solution can be prepared simply by adding a sample.
  • An all-in-one composition is exemplified.
  • the kit of the present invention may contain a thermostable enzyme having reverse transcriptase activity and DNA-dependent DNA polymerase activity instead of the combination of thermostable reverse transcriptase and thermostable DNA polymerase.
  • RT-PCR was carried out in a final volume of 25 ⁇ L reaction solution using a kit of RR296A TaKaRa Norovirus GI / GII detection kit (for high-speed detection) (manufactured by Takara Bio Inc.) according to the description in the instruction manual.
  • the RT-PCR conditions were as follows: treatment at 42 ° C. for 5 min and 94 ° C. for 30 sec, followed by 40 cycles of 94 ° C. for 5 sec-56 ° C. for 30 sec and one cycle for 90 ° C. for 5 sec-56 ° C. for 30 sec.
  • TP900 ThermalCycler Dice registered trademark
  • RealTime System II manufactured by Takara Bio Inc.
  • the amplified product was detected under any conditions, and it was confirmed that sufficient RNA for RT-PCR was obtained particularly under heating conditions of 70 ° C. or higher. Therefore, it was suggested that if the heat resistance of the reverse transcriptase is 70 ° C. or higher, the RNA virus can be destroyed in the reverse transcription reaction solution, and a reverse transcription reaction that does not require opening of the reaction vessel can be suitably performed.
  • Reference Example 2 Examination with stool sample Human stool from which norovirus GI was detected by a conventional method was examined. That is, the stool was suspended in PBS to about 10% (w / v), and a supernatant obtained by centrifugation was prepared. The virus solution was subjected to RT-PCR by the method described in Reference Example 1. The results are shown in Table 2.
  • the amplified product was detected under any conditions, and it was confirmed that sufficient RNA for RT-PCR was obtained particularly under heating conditions of 70 ° C. or higher. Therefore, as in Reference Example 1, if the heat resistance of the reverse transcriptase is 70 ° C. or higher, the RNA virus is destroyed in the reverse transcription reaction solution, and a reverse transcription reaction that does not require opening of the reaction vessel is suitably performed. It was suggested that it can be done.
  • Example 1 Study in One-Tube Reaction System Using the virus solution prepared in Reference Example 1, the target gene amplification in a solution containing all reagents necessary for reverse transcription reaction and DNA amplification reaction was examined. That is, the virus solution having a volume of 1/25 of the reaction solution, 5 ⁇ RT-PCR buffer attached to R510A TaKaRa Tth (manufactured by Hojin Medical Biotechnology Co., Ltd.), final concentration of 2.5 mM manganese acetate, final concentration is Norovirus G1 gene amplification primer pair having the base sequences described in SEQ ID NOS: 1 and 2 in the sequence table of 0.0004 mM, and G1 gene detection having the base sequences described in SEQ ID NOS: 3 and 4 in the sequence table at a final concentration of 0.0002 mM A total amount of 25 ⁇ L containing a probe for use, final concentration of 0.3 mM dNTP, 5 U of Tth DNA polymerase (manufactured by Takahashi Medical Bio
  • the surfactant was selected from Tween (registered trademark) 20, Triton (registered trademark table) X-100, Nonidet (registered trademark) P-40, poly (ethyleneglycol) 4-nonylphenyl 3-sulfopropyl ether.
  • a non-surfactant was also prepared as a control.
  • the final concentrations of various surfactants were set to 0.02, 0.2, 2, 4, 6, 8, and 10% (V / V).
  • the RT-PCR conditions were a 5-cycle reaction in which 90 ° C. for 3 min, 58 ° C. for 30 min, and 95 ° C. for 30 sec, and a cycle of 95 ° C. for 5 sec-56 ° C.
  • the same apparatus as the reference example 1 was used for the nucleic acid amplifier.
  • the target nucleic acid was amplified even when the surfactant was not used, but the Ct value was smaller when either surfactant was used than when the surfactant was not used.
  • the Ct value was reduced by 4 or more when the final concentration was 2% (V / V) or more regardless of the type of the surfactant.
  • RNA virus processing method of the present invention the suspension supernatant containing RNA virus is added to the solution containing all the reagents necessary for reverse transcription reaction and DNA amplification reaction in One-Tube. It is confirmed that reverse transcription reaction and DNA amplification reaction can be continuously performed in the same container, and that the sensitivity is increased up to 16 times by combining the surfactant with the method of the present invention. did it.
  • Example 2 Examination of One-Tube Reaction System Using Fecal Specimen The detection of norovirus in a real specimen was examined in a solution containing all the reagents necessary for reverse transcription reaction and DNA amplification reaction. That is, a norovirus-positive stool specimen collected from a subject with informed consent was suspended in PBS so as to be about 10% (w / v), and then centrifuged at 15,000 rpm for 5 minutes. . 1 ⁇ L of the supernatant thus obtained was used for the following reaction.
  • Tth DNA polymerase manufactured by Hojin Medical Biotechnology Co., Ltd.
  • Tth DNA polymerase variant described in Japanese Patent No. 3844975
  • This mutant has been reported to have high reverse transcriptase activity efficiency, and position 681 of the amino acid sequence described in Taq DNA polymerase (Genbank Acc. No. BAA06775.1) is Genbank Acc. No. Since the amino acid sequence of Tth DNA polymerase based on the amino acid sequence described in WP_011228405 corresponds to position 683, a Tth DNA polymerase mutant (E683K) in which glutamic acid at position 683 of the amino acid sequence was substituted with lysine was prepared.
  • an artificial gene having a sequence in which a mutation was introduced at a specific site in the amino acid sequence was chemically synthesized.
  • the obtained artificial gene was introduced into plasmid pET6xHN-N (Takara Bio USA) using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio USA).
  • Escherichia coli was transformed with the plasmid, and seed culture and main culture were performed according to a conventional method. After completion of the culture, the cells were collected, and after disrupting the cells, a crude enzyme solution was prepared. The obtained crude enzyme solution was used as a Tth DNA polymerase mutant crude purification solution for the following tests.
  • the primer pair used is “Norovirus detection method” notified by the Ministry of Health, Labor and Welfare, Food Safety Department, Food Safety Department, Monitoring and Safety Division (Approved by Food Safety Supervision No. 1105001 dated November 5, 2003, final revision: 2013) It is a primer for GI detection and GII detection having the same base sequence as described in Food Safety Supervision No. 1022 No. 1) (hereinafter referred to as “official method”) dated October 22,
  • RT-PCR conditions for Tth DNA polymerase are 5 cycles of 90 ° C 3 minutes, 58 ° C 5 minutes or 30 minutes, 95 ° C 30 seconds, then 95 ° C 5 seconds, 56 ° C 30 seconds.
  • the reaction was set to a 40-cycle reaction with 90 ° C. for 5 seconds and 56 ° C. for 30 seconds as one cycle.
  • a 5-cycle reaction consisting of 95 ° C. for 5 seconds and 56 ° C. for 30 seconds, 90 ° C. continuously
  • the reaction was set to 40 cycles with 5 seconds at 56 ° C. and 30 seconds as one cycle.
  • the thermal cycler performed real-time PCR using TP990 ThermalCycler Dice (registered trademark) RealTime System III (manufactured by Takara Bio Inc.), and measured the Ct value.
  • the RNA virus treatment method of the present invention can be used for stool specimens, and can be used for both normal RT-PCR methods and RT-PCR methods with a short reverse transcription reaction time. Was confirmed.
  • Example 3 Confirmation of the effect of low-concentration surfactant If a large amount of surfactant is present in the solution, the risk of foaming may occur. In particular, in the case of mass processing using an automatic dispensing device, there is a possibility of dispensing error, so whether or not the effect obtained by adding the surfactant confirmed in Example 1 can be obtained at a lower concentration is referred. Further verification was performed using the virus solution prepared in Example 1. Verification was performed using Nonidet® P-40. Similar to Example 1, those without surfactant and those with final surfactant concentrations of 0.02, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, Reaction liquids of 1.4, 1.6, and 1.8% (V / V) were prepared.
  • the PCR reaction composition other than the surfactant concentration is the same as in Example 1.
  • RT-PCR conditions are as follows: 90 ° C. for 3 minutes, 58 ° C. for 30 minutes, 95 ° C. for 30 seconds, and 95 ° C. for 5 seconds to 56 ° C. for 30 seconds without opening the reaction vessel cover 5
  • a cycle reaction was carried out, and a 40-cycle reaction was carried out with 90 ° C. for 5 seconds-56 ° C. for 30 seconds as one cycle.
  • the nucleic acid amplification apparatus performed real-time PCR using the same apparatus as what was used in Example 2, and measured Ct value. The results are shown in Table 3-1 and Table 3-2.
  • Example 4 Confirmation of the effect of the present invention
  • the target gene in the solution containing all of the virus solution prepared in Reference Example 1 and the reagents necessary for the reverse transcription reaction and the DNA amplification reaction. Amplification was examined.
  • NV Primer / Probe Mix7 (manufactured by Takara Bio Inc.) contained in the component 3 was added to prepare a reaction solution A having a final volume of 25 ⁇ L.
  • 1 ⁇ L of the virus solution, 5 ⁇ Tth RT-PCR buffer solution (manufactured by Takahashi Medical Biotechnology Co., Ltd.) contained in the components of R510A TaKaRa Tth, final concentration 2.5 mM manganese acetate, final concentration 0.3 mM DNTP, final concentration 0.2 U / ⁇ L of rTth DNA Polymerase (TTH-301 rTth DNA Polymerase manufactured by Toyobo Co., Ltd.), primer and probe as RR298A TaKaRa Norovirus GI / GII detection kit Ver.
  • NV Primer / Probe Mix7 (manufactured by Takara Bio Inc.) 8 ⁇ L contained in 3 components, and a final volume of 25 ⁇ L containing 1,3 propanediol having a final concentration of 4% and L-carnitine having a final concentration of 0.3 M as additives.
  • Reaction solution B was prepared.
  • reaction solutions C and D were prepared in which norovirus synthetic RNA solution was added instead of the virus solution in the reaction solutions A and B.
  • the norovirus synthetic RNA solution was obtained from Microbiologicals TM Helix Elite TM Norobirus GII.
  • RNA (Microbiologics Inc.) lyophilized to a 10 ⁇ 7 square copy of diluted 10-fold with further H 2 O stock solution of 10 ⁇ 8 square copy made by dissolving with H 2 O that comes RNA It was a solution.
  • reaction solutions A to D were subjected to normal RT-PCR without a high temperature heating step. Separately, the reaction solution A was subjected to PCR after high-temperature reverse transcription.
  • RT-PCR conditions are: high temperature reverse transcription reaction, 90 ° C for 3 minutes, 50 ° C for 30 minutes reverse transcription, 95 ° C for 30 seconds heat denaturation, 95 ° C for 5 seconds, 56 ° C for 60 seconds for one cycle.
  • a 5-cycle reaction was set to a 40-cycle reaction with one cycle of 90 ° C. for 5 seconds and 56 ° C. for 60 seconds.
  • the high temperature reverse transcription reaction at 90 ° C. for 3 minutes was omitted, and after reverse transcription at 50 ° C. for 30 minutes, the same PCR conditions were used.
  • the nucleic acid amplification apparatus was subjected to real-time PCR using the same apparatus as that used in Example 2, and the Ct value was measured. The results are shown in Table 4.
  • the present invention is capable of highly efficiently eluting RNA from the capsid of RNA virus by holding the sample at a high temperature in the RT-PCR reaction solution, and it is necessary to open the reaction vessel. It was revealed that the reverse transcription reaction that is not performed can be suitably performed.
  • the present invention is used for food hygiene inspection, environmental inspection, clinical diagnosis and the like.

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Abstract

La présente invention concerne un procédé de traitement de virus à ARN qui comprend : (a) une étape dans laquelle un échantillon comprenant éventuellement un virus à ARN est préparé ; (b) une étape dans laquelle un liquide de réaction de transcription inverse comprenant l'échantillon provenant de l'étape (a) et une enzyme de transcription inverse résistante à la chaleur est préparée ; et (c) une étape dans laquelle le liquide de réaction obtenu à l'étape (b) est chauffé.
PCT/JP2019/017490 2018-05-02 2019-04-24 Procédé de traitement de virus à arn Ceased WO2019212023A1 (fr)

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