WO2018038088A1 - Procédé d'amplification et de détection d'acide nucléique, et solution d'amplification et de détection d'acide nucléique - Google Patents

Procédé d'amplification et de détection d'acide nucléique, et solution d'amplification et de détection d'acide nucléique Download PDF

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WO2018038088A1
WO2018038088A1 PCT/JP2017/029891 JP2017029891W WO2018038088A1 WO 2018038088 A1 WO2018038088 A1 WO 2018038088A1 JP 2017029891 W JP2017029891 W JP 2017029891W WO 2018038088 A1 WO2018038088 A1 WO 2018038088A1
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nucleic acid
acid amplification
reaction solution
pcr reaction
hybridization
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Japanese (ja)
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弘輝 道志
聡史 古川
淳憲 一色
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東洋製罐グループホールディングス株式会社
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to amplification and detection of nucleic acid contained in a biological sample.
  • nucleic acid detection methods a target region is amplified by a nucleic acid amplification method such as PCR (polymerase chain reaction), and the resulting nucleic acid amplification product is detected using a DNA chip (DNA microarray) or the like. It has been known.
  • a nucleic acid amplification method such as PCR (polymerase chain reaction)
  • DNA chip DNA microarray
  • a nucleic acid having a base sequence complementary to a nucleic acid amplification product (hereinafter sometimes referred to as a probe) is immobilized in advance on a DNA chip, and a PCR reaction solution containing a nucleic acid amplification product obtained by PCR Is dropped onto a DNA chip, and hybridization is performed to bind the nucleic acid amplification product to the probe, whereby the nucleic acid to be detected is detected.
  • a hybridization buffer is mixed with the PCR reaction solution, and the obtained mixture solution is dropped onto the DNA chip to add the DNA chip.
  • the cover was put on and hybridization was performed.
  • Such buffer preparation and mixing operations are not only complicated, but there are also problems that bubbles may be mixed into the mixed solution to inhibit hybridization, or a test error may occur due to forgetting to add a buffer.
  • the operation speed can be improved, and bubbles can be prevented from being mixed. Further, it is possible to prevent an inspection error due to forgetting to add the buffer, and to obtain an effect that the nucleic acid amplification product is not diluted by the buffer. Furthermore, if the PCR reaction solution containing the nucleic acid amplification product can be directly dropped onto the DNA chip as it is and hybridization can be performed, the inspection process can be simplified.
  • the salt concentration (cation concentration) of the solution dropped on the DNA chip is preferably higher than that of a normal PCR reaction solution. Therefore, conventionally, hybridization has been generally facilitated by adding a hybridization buffer to a PCR reaction solution to increase the salt concentration and then dropping it onto a DNA chip.
  • a high salt concentration is preferable in the hybridization is that the phosphate group in the DNA molecule is negatively charged, and the two strands of DNA become unstable by repelling each other.
  • the cation concentration of the solution used for the above is high, the negative charge is shielded, the double strand is stabilized, and the detection accuracy is improved.
  • PCR if PCR is performed using a mixed solution previously mixed with a hybridization buffer prior to PCR, PCR does not proceed properly.
  • the hybridization buffer has a high salt concentration and generally contains a surfactant.
  • the PCR reaction solution has a high salt concentration, and the surface activity is increased. This is because when the agent is mixed, the activity of the nucleic acid synthase is inhibited and the nucleic acid amplification reaction cannot be performed appropriately.
  • the present inventors have diligently studied, and by mixing a specific substance in the PCR reaction solution, PCR can be appropriately performed, and the PCR reaction solution containing the obtained nucleic acid amplification product is used as it is, The present invention was completed by successfully performing hybridization without preparing and mixing a hybridization buffer.
  • PCR can be appropriately performed using the PCR reaction solution, and the PCR reaction containing the obtained nucleic acid amplification product By using the solution as it was, hybridization could be performed sufficiently.
  • a technique using a metal salt and / or a water-soluble polymer a PCR nucleic acid amplification product purification method described in Patent Document 1 or a DNA analysis method described in Patent Document 2
  • the biological sample storage method can be mentioned.
  • a technique using a metal salt and / or a water-soluble polymer in relation to hybridization a method for preparing a hybridization solution described in Patent Document 3 can be mentioned.
  • a nucleic acid amplification reaction is performed, and the reaction solution containing the obtained nucleic acid amplification product is used as it is for hybridization. It did not make it possible to do enough.
  • the present invention has been made in view of the above circumstances, and a nucleic acid amplification and detection method capable of performing hybridization using a reaction solution containing a nucleic acid amplification product obtained as it is after a nucleic acid amplification reaction by PCR or the like, and a nucleic acid
  • the purpose is to provide a solution for amplification and detection.
  • the nucleic acid amplification and detection method of the present invention amplifies a target region in a nucleic acid contained in a sample by a nucleic acid amplification reaction, and the obtained nucleic acid amplification product is complementary to the nucleic acid amplification product.
  • a nucleic acid amplification reaction is performed using the reaction solution containing the nucleic acid amplification product obtained by the nucleic acid amplification reaction as it is.
  • the nucleic acid amplification and detection solution of the present invention amplifies a target region in a nucleic acid contained in a sample by a nucleic acid amplification reaction, and the obtained nucleic acid amplification product has a base sequence complementary to the nucleic acid amplification product.
  • the present invention after the nucleic acid amplification reaction by PCR or the like, it is possible to carry out hybridization using the reaction solution containing the obtained nucleic acid amplification product as it is.
  • FIG. 1 shows a detection result of a nucleic acid amplification product obtained by a nucleic acid amplification and detection method according to an embodiment of the present invention using a DNA chip (when NaCl is added to a PCR reaction solution).
  • FIG. 2 shows a detection result of a nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip (when KCl is added to a PCR reaction solution).
  • FIG. 3 shows a detection result (when MgCl 2 is added to a PCR reaction solution) of a nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip.
  • FIG. 1 shows a detection result of a nucleic acid amplification product obtained by a nucleic acid amplification and detection method according to an embodiment of the present invention using a DNA chip (when NaCl is added to a PCR reaction solution).
  • FIG. 4 shows the detection result (when Na 2 SO 4 is added to the PCR reaction solution) of the nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip.
  • FIG. 5 shows a detection result of a nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip (when CH 3 COONa is added to a PCR reaction solution).
  • FIG. 6 shows a detection result (when PEG 6000 is added to a PCR reaction solution) of a nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip.
  • FIG. 5 shows a detection result of a nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip (when CH 3 COONa is added to a PCR reaction solution).
  • FIG. 7 shows the detection results (when various water-soluble polymers are added to the PCR reaction solution) of the nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention.
  • FIG. 8 shows a detection result (when NaCl and PEG 6000 are added to the PCR reaction solution) of the nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention.
  • FIG. 9 shows the detection result of the nucleic acid amplification product obtained by the nucleic acid amplification and detection method according to the embodiment of the present invention using a DNA chip (when NaCl and various water-soluble polymers are added to the PCR reaction solution).
  • a target region in a nucleic acid contained in a sample is amplified by a nucleic acid amplification reaction, and the obtained nucleic acid amplification product is combined with a nucleic acid having a base sequence complementary to the nucleic acid amplification product.
  • a nucleic acid amplification and detection method for detecting by performing hybridization wherein a reaction solution for nucleic acid amplification contains a metal salt and / or a water-soluble polymer, and a nucleic acid amplification reaction is performed using the reaction solution
  • Hybridization is performed using the reaction solution as it is without adding a buffer to the reaction solution containing the nucleic acid amplification product obtained by the nucleic acid amplification reaction.
  • a nucleic acid amplification step for amplifying a target region in a nucleic acid contained in a sample by PCR or the like, and a nucleic acid having a base sequence complementary to the obtained nucleic acid amplification product
  • a hybridization step for detecting by binding.
  • the reaction solution contains a metal salt and / or a water-soluble polymer, and the reaction solution is used to perform a nucleic acid amplification reaction.
  • the hybridization step the reaction solution is used as it is for hybridization. Do.
  • the nucleic acid amplification step can be performed, for example, as follows. First, genomic DNA is extracted from a biological sample. Genomic DNA can be extracted by a general method such as a method using a CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction apparatus. Next, a specific gene or the like in the extracted genomic DNA is used as a target region and amplified by a PCR method or the like to obtain a nucleic acid amplification product.
  • CTAB method Cetyl trimethyl ammonium bromide
  • the nucleic acid amplification method is not limited to the PCR method, and other methods such as RT-PCR, NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification), and LCR (ligase chain reaction) may be used. good.
  • a target region is amplified using a PCR reaction solution containing a primer set for amplifying the target region.
  • a general thermal cycler or the like can be used as the PCR apparatus.
  • the PCR reaction can be performed, for example, under the following reaction conditions.
  • the PCR reaction solution contains a metal salt and / or a water-soluble polymer.
  • the PCR reaction solution includes, for example, a nucleic acid synthesis substrate (dNTPmixture (dCTP, dATP, dTTP, dGTP)), a primer set, a nucleic acid synthase (EX Taq HotStart DNA polymerase, etc. ), Genomic DNA of the sample, buffer solution, and those containing water as the remaining components can be suitably used.
  • the nucleic acid amplification and detection solution of the present embodiment amplifies a target region in a nucleic acid contained in a sample by a nucleic acid amplification reaction, and the obtained nucleic acid amplification product is a nucleic acid having a base sequence complementary to the nucleic acid amplification product.
  • the reaction solution is characterized by containing a metal salt and / or a water-soluble polymer.
  • the nucleic acid amplification and detection solution of the present embodiment corresponds to a nucleic acid amplification reaction solution containing a metal salt and / or a water-soluble polymer used in the nucleic acid amplification and detection method of the present embodiment.
  • the metal salt for example, NaCl, KCl, MgCl 2 , Na 2 SO 4 , CH 3 COONa, or the like can be suitably used. .
  • a nonionic water-soluble polymer can be suitably used as the water-soluble polymer.
  • the nonionic water-soluble polymer polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and derivatives thereof can be suitably used.
  • the polyethylene glycol derivative include Tween surfactant (a kind of nonionic surfactant, polyoxyethylene sorbitan fatty acid ester) and Triton surfactant (a kind of nonionic surfactant, polyoxyethylene). Alkylphenyl ether) and the like.
  • the primer set to be contained in the PCR reaction solution is not particularly limited, and the target region in the nucleic acid contained in the sample is determined.
  • a primer set consisting of a forward primer and a reverse primer for amplification by PCR is used. If there are multiple target regions, each corresponding primer set can be used in one PCR reaction or in a separate PCR reaction.
  • the fluorescent primer Cy5 can be used to label the 5 ′ end of the reverse primer and / or forward primer.
  • the hybridization step can be performed, for example, as follows. That is, a PCR reaction solution containing a nucleic acid amplification product obtained by PCR is dropped onto a DNA chip or the like on which a probe having a base sequence complementary to the nucleic acid amplification product is immobilized, and the nucleic acid amplification product is used as a probe. Combine. At this time, in the nucleic acid amplification and detection method of the present embodiment, the PCR reaction solution containing the nucleic acid amplification product is used as it is for the hybridization, and a buffer is separately prepared for the hybridization as in the conventional method. Do not mix with the PCR reaction solution.
  • hybridization is not limited to the method using a DNA chip, but other methods such as Southern hybridization and Northern hybridization may be used.
  • a probe having a base sequence complementary to a nucleic acid amplification product obtained by amplifying a target region in the DNA of the organism to be examined is immobilized on a DNA chip in advance.
  • the probe can be synthesized by a general DNA synthesizer.
  • an affixed type DNA chip is prepared as a DNA chip, it can be prepared by fixing a probe on a glass substrate with a DNA spotter and forming spots corresponding to each probe.
  • a synthetic DNA chip is produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique.
  • the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used.
  • the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .
  • the presence of the nucleic acid amplification product is confirmed by dropping the reaction solution containing the nucleic acid amplification product directly onto the DNA chip and detecting the fluorescent label of the nucleic acid amplification product bound to the probe immobilized on the DNA chip. . This makes it possible to determine the presence or absence of the organism to be examined in the sample.
  • the detection of the fluorescent label can be performed using a general label detection device such as a fluorescence scanning device.
  • a fluorescence scanning device For example, the fluorescence intensity of the nucleic acid amplification product can be measured using a GENOGATE (registered trademark) reader of Toyo Seikan Group Holdings, Inc. This can be done by measuring.
  • the measurement result can be acquired as a median value of fluorescence intensity. It can also be obtained as an S / N ratio (Signal to Noise ratio) value.
  • the label is not limited to fluorescence, and other labels can also be used.
  • the reaction solution containing the nucleic acid amplification product can be used as it is for hybridization.
  • the work of preparing and mixing with the reaction solution can be omitted, and the working speed can be improved.
  • the reaction solution containing the nucleic acid amplification product can be directly dropped onto the DNA chip as it is and hybridization can be performed, the inspection process can be simplified.
  • nucleic acid amplification and detection method of the present invention and the tests conducted for confirming the effects of the nucleic acid amplification and detection solution will be specifically described.
  • Test 1 A nucleic acid amplification reaction by PCR was performed under the conditions shown below, and a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip to confirm whether hybridization was sufficiently performed. Specifically, it was performed as follows.
  • RIMD 05091053 (serotype O157: H7), an Escherichia coli strain obtained from Research Institute for Microbial Diseases (RIMD), Osaka University, was used.
  • Nutrient broth (Nippon Becton Dickyson Co., Ltd.) was poured into the ampoule containing the Escherichia coli strain, and suspended by a gentle pipetting operation until a uniform bacterial solution was obtained.
  • the obtained bacterial suspension was smeared on a heart infusion agar medium (Nissui Pharmaceutical Co., Ltd.) and aerobically cultured overnight at 37 ° C. to regenerate and enrich the bacteria.
  • the colonies of the enriched bacteria were collected with a platinum ear and used as a cell for DNA extraction.
  • DNA was extracted from the cells for DNA extraction using DNeasy Blood & Tissue Kit (Qiagen Co., Ltd.) according to the method for extracting Gram-negative bacteria described in the manual. This DNA extract was used as a template for PCR.
  • vtx1 gene was amplified by PCR method.
  • a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 were used and included in the PCR reaction solution.
  • This primer set used was synthesized by Sigma-Aldrich Japan LLC. The 5 'end of the reverse primer was fluorescently labeled with Cy5.
  • Ex Taq Buffer manufactured by Takara Bio Inc.
  • NaCl manufactured by Wako Pure Chemical Industries, Ltd.
  • the following composition is prepared. Created. There were six NaCl concentrations of 0 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 60 mM, and these six types of PCR reaction solutions were prepared, and nucleic acid amplification reaction by PCR was performed for each.
  • DNA was amplified by the nucleic acid amplification apparatus (Thermal Cycler Ep Gradient Eppendorf Co., Ltd.) under the following conditions.
  • Gene Silicon registered trademark
  • SEQ ID NO: 3 The probe used was synthesized by Life Technologies Japan.
  • the probe was immobilized on the substrate using a microarrayer. This probe is selected from the vtx1 gene region in the DNA of the organism to be detected, and can be complementarily bound to the nucleic acid amplification product obtained by PCR.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a NaCl concentration of 0 mM (no addition of metal salt), 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, and mixing the hybridization buffer with the PCR reaction Hybridization was performed without any.
  • PCR reaction solution with a NaCl concentration of 0 ⁇ m, separately add 2 ⁇ l of hybridization buffer (3 ⁇ SSC citrate-saline + 0.3% SDS) to 4 ⁇ l of the PCR reaction solution containing the nucleic acid amplification product. The mixture was added dropwise to the DNA chip and allowed to stand at 45 ° C. for 1 hour for hybridization.
  • hybridization buffer 3 ⁇ SSC citrate-saline + 0.3% SDS
  • the PCR reaction solution containing the nucleic acid amplification product obtained by adding NaCl to the PCR reaction solution (the concentration of NaCl is 25 mM, 30 mM, 40 mM, 50 mM)
  • the concentration of NaCl is 25 mM, 30 mM, 40 mM, 50 mM
  • a fluorescence intensity was obtained, and compared with a PCR reaction solution with a NaCl concentration of 0 mM and a buffer mixture. It can be seen that a fluorescence intensity comparable to that obtained is obtained. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently performed by adding the metal salt and using the reaction solution containing the nucleic acid amplification product as it was.
  • Test 2 The PCR reaction solution was subjected to a nucleic acid amplification reaction by PCR under the same conditions as in Test 1, except that KCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added at various concentrations instead of NaCl as a metal salt. A PCR reaction solution containing the obtained nucleic acid amplification product was dropped onto a DNA chip to confirm whether hybridization was sufficiently performed.
  • KCl manufactured by Wako Pure Chemical Industries, Ltd.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a KCl concentration of 0 mM (without addition of metal salt), 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, and mixing the hybridization buffer with the PCR reaction Hybridization was performed without any.
  • PCR reaction solution with a KCl concentration of 0 ⁇ m, separately add 2 ⁇ l of hybridization buffer (3 ⁇ SSC citrate-saline + 0.3% SDS) to 4 ⁇ l of the PCR reaction solution containing the nucleic acid amplification product. The mixture was added dropwise to the DNA chip and allowed to stand at 45 ° C. for 1 hour for hybridization.
  • hybridization buffer 3 ⁇ SSC citrate-saline + 0.3% SDS
  • the PCR reaction solution containing KCl in the PCR reaction solution and the nucleic acid amplification product obtained by amplification (KCl concentrations of 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ mM)
  • KCl concentrations of 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ mM KCl concentrations of 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ mM
  • excellent fluorescence intensity was obtained, and compared with a PCR reaction solution with a KCl concentration of 0 mM and a buffer mixed. It can be seen that a fluorescence intensity comparable to that obtained is obtained. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently performed by adding the metal salt and using the reaction solution containing the nucleic acid amplification product as it was.
  • Test 3 A PCR reaction solution was subjected to a nucleic acid amplification reaction by PCR under the same conditions as in Test 1, except that MgCl 2 (manufactured by Life Technologies Japan Co., Ltd.) was added at various concentrations instead of NaCl as a metal salt. A PCR reaction solution containing the obtained nucleic acid amplification product was dropped onto a DNA chip to confirm whether hybridization was sufficiently performed.
  • MgCl 2 manufactured by Life Technologies Japan Co., Ltd.
  • PCR reaction solution 0 mM, 5 mM, 10 mM, and 15 mM. These four types of PCR reaction solutions were prepared, and nucleic acid amplification reaction by PCR was performed on each of them.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a MgCl 2 concentration of 0 mM (no addition of metal salt), 5 mM, 10 mM, or 15 mM, hybridization without mixing the hybridization buffer into the PCR reaction solution Went.
  • PCR reaction solution having a MgCl 2 concentration of 0 mM
  • 4 ⁇ l of the PCR reaction solution containing the nucleic acid amplification product and 2 ⁇ l of hybridization buffer (3 ⁇ SSC citrate-saline + 0.3% SDS) Were added dropwise to a DNA chip and allowed to stand at 45 ° C. for 1 hour for hybridization.
  • the PCR reaction solution was containing MgCl 2, the PCR reaction solution containing the obtained nucleic acid amplification products by amplifying (concentration of MgCl 2 is 5 mM, 10 mM), the concentration of MgCl 2 Compared to the PCR reaction solution with 0 mM and no buffer mixed, excellent fluorescence intensity was obtained, and compared with the PCR reaction solution with MgCl 2 concentration of 0 mM and buffer mixed, It can be seen that the fluorescence intensity without any is obtained. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently performed by adding the metal salt and using the reaction solution containing the nucleic acid amplification product as it was.
  • Test 4 The PCR reaction solution was subjected to a nucleic acid amplification reaction by PCR under the same conditions as in Test 1, except that Na 2 SO 4 (manufactured by Wako Pure Chemical Industries, Ltd.) was added at various concentrations instead of NaCl as a metal salt. Then, a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip to confirm whether or not hybridization was sufficiently performed.
  • Na 2 SO 4 manufactured by Wako Pure Chemical Industries, Ltd.
  • PCR reaction solution There were four concentrations of Na 2 SO 4 in the PCR reaction solution: 0 mM, 5 mM, 10 mM, and 15 mM. These four types of PCR reaction solutions were prepared, and nucleic acid amplification reaction by PCR was performed on each of them. .
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a Na 2 SO 4 concentration of 0 mM (without addition of a metal salt), 5 mM, 10 mM, or 15 mM without mixing a hybridization buffer with the PCR reaction solution, Hybridization was performed.
  • the PCR reaction solution is made to contain the Na 2 SO 4
  • the PCR reaction solution containing the obtained nucleic acid amplification products by amplifying the (concentration of Na 2 SO 4 is 10 mM, 15 mM) is although Na 2 concentration of SO 4 is when compared to the PCR reaction solution was mixed with buffer fluorescence intensity is low at 0 mM, higher fluorescence intensity than the PCR reaction solution in which the concentration of Na 2 SO 4 is not mixed with buffer at 0 mM
  • Test 5 The PCR reaction solution was subjected to a nucleic acid amplification reaction by PCR under the same conditions as in Test 1, except that CH 3 COONa (manufactured by Wako Pure Chemical Industries, Ltd.) was added at various concentrations instead of NaCl as a metal salt. Then, a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip, and it was confirmed whether or not hybridization was sufficiently performed.
  • CH 3 COONa manufactured by Wako Pure Chemical Industries, Ltd.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a CH 3 COONa concentration of 0 mM (no addition of metal salt), 20 mM, and 50 mM, hybridization was performed without mixing the hybridization buffer with the PCR reaction solution. It was.
  • Test 6 Nucleic acid amplification reaction by PCR under the same conditions as in Test 1 except that PEG6000 (manufactured by Wako Pure Chemical Industries, Ltd.) was added as a water-soluble polymer at various concentrations without adding a metal salt to the PCR reaction solution. Then, a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip to confirm whether or not hybridization was sufficiently performed.
  • PEG6000 manufactured by Wako Pure Chemical Industries, Ltd.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, using a PCR reaction solution with a PEG 6000 concentration of 0 mM (no addition of water-soluble polymer), 5% (w / v), and 10% (w / v), a hybridization buffer is added to the PCR reaction solution. Hybridization was performed without mixing.
  • PCR reaction solution with a PEG6000 concentration of 0 ⁇ mM
  • hybridization buffer 3 ⁇ SSC citrate-saline solution + 0.3% SDS
  • the mixture was added dropwise to the DNA chip and allowed to stand at 45 ° C. for 1 hour for hybridization.
  • the concentration of PEG 6000 is 5% (w / v)
  • the concentration of PEG 6000 is 5% (w / v)
  • the fluorescence intensity is lower than that of the PCR reaction solution mixed with the buffer at 0 ⁇ mM
  • the fluorescence intensity higher than that of the PCR reaction solution with the PEG6000 concentration of 0 ⁇ mM and no buffer mixed is obtained. That is, in this PCR reaction solution, it can be seen that hybridization was sufficiently performed by adding the water-soluble polymer and using the reaction solution containing the nucleic acid amplification product as it was.
  • Test 7 A PCR reaction containing a nucleic acid amplification product obtained by performing a nucleic acid amplification reaction by PCR under the same conditions as in Test 1 except that various water-soluble polymers were added to the PCR reaction solution without adding a metal salt. The solution was dropped on the DNA chip to confirm whether hybridization was sufficiently performed.
  • water-soluble polymer PEG 6000 (manufactured by Wako Pure Chemical Industries, Ltd.), PEG 20000 (manufactured by Wako Pure Chemical Industries, Ltd.), PVP (manufactured by Kishida Chemical Co., Ltd.), and Tween 20 (manufactured by MP Bio Japan) are used.
  • the final concentration was 5% (w / v).
  • a PCR reaction solution to which no water-soluble polymer was added was also prepared. And each performed nucleic acid amplification reaction by PCR.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, a PCR reaction solution without addition of a water-soluble polymer, the concentration of PEG 6000 is 5% (w / v), the concentration of PEG 20000 is 5% (w / v), the concentration of PVP is 5% (w / v), Tween 20 Hybridization was performed using a PCR reaction solution having a concentration of 5% by weight (w / v) without mixing a hybridization buffer with the PCR reaction solution.
  • hybridization buffer 3 ⁇ SSC citrate-saline + 0.3% SDS
  • PCR reaction solution containing nucleic acid amplification products For PCR reaction solution without addition of water-soluble polymer, separately add 2 ⁇ l of hybridization buffer (3 ⁇ SSC citrate-saline + 0.3% SDS) to 4 ⁇ l of PCR reaction solution containing nucleic acid amplification products. The mixture was added dropwise to the DNA chip and allowed to stand at 45 ° C. for 1 hour for hybridization.
  • the buffer was mixed without adding the water-soluble polymer.
  • the fluorescence intensity was lower than that of the PCR reaction solution, it was higher than that of the PCR reaction solution in which no water-soluble polymer was added and no buffer was mixed, and the fluorescence intensity was improved from nearly 10% to nearly 30%. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently performed by adding the water-soluble polymer and using the reaction solution containing the nucleic acid amplification product as it was.
  • Test 8 This test was performed to confirm the effect of adding both a metal salt and a water-soluble polymer to the PCR reaction solution. Specifically, a nucleic acid amplification reaction by PCR under the same conditions as in Test 1 except that a PCR reaction solution to which a metal salt was added and a PCR reaction solution to which both a metal salt and a water-soluble polymer were added were prepared. Then, a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip to confirm whether or not hybridization was sufficiently performed.
  • the metal salt 40 mM NaCl was used.
  • the water-soluble polymer PEG 6000 was used, and in the PCR reaction solution, the final concentration was 0% (w / v), 5% (w / v), and 10% (w / v). .
  • a PCR reaction solution to which neither metal salt nor water-soluble polymer was added was also prepared. And each performed nucleic acid amplification reaction by PCR.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, a PCR reaction solution to which neither a metal salt nor a water-soluble polymer was added, and a metal salt was added, and the concentration of PEG 6000 was 0% (w / v), 5% (w / v), 10% ( Hybridization was performed using the PCR reaction solution of w / v) without mixing the hybridization buffer with the PCR reaction solution.
  • both the metal salt and the water-soluble polymer were used for the PCR reaction solution containing the nucleic acid amplification product obtained by adding and amplifying both the metal salt and the water-soluble polymer in the PCR reaction solution. It can be seen that an excellent fluorescence intensity is obtained as compared with a PCR reaction solution in which a buffer is mixed without adding. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently carried out using the reaction solution containing the nucleic acid amplification product as it was by adding a metal salt and a water-soluble polymer.
  • Test 9 This test was performed in order to confirm the effects when both a metal salt and a water-soluble polymer were added to the PCR reaction solution and various water-soluble polymers were used. Specifically, a nucleic acid amplification reaction by PCR under the same conditions as in Test 1 except that a PCR reaction solution to which a metal salt was added and a PCR reaction solution to which a metal salt and various water-soluble polymers were added were prepared. Then, a PCR reaction solution containing the obtained nucleic acid amplification product was dropped on the DNA chip to confirm whether or not hybridization was sufficiently performed.
  • the metal salt 40 mM NaCl was used.
  • the water-soluble polymer examples include PEG600 (manufactured by GL Sciences Inc.), PEG1000 (manufactured by GL Sciences Inc.), PEG6000 (manufactured by Wako Pure Chemical Industries, Ltd.), PEG12000 (manufactured by Sigma Aldrich Japan LLC), PEG20000 ( Wako Pure Chemical Industries, Ltd.), PVP (Kishida Chemical Co., Ltd.), Tween 20 (MP Bio Japan Co., Ltd.), Tween 80 (Wako Pure Chemical Industries, Ltd.) are used, and each is used in each PCR reaction solution. The final concentration was 5% (w / v).
  • a PCR reaction solution to which neither metal salt nor water-soluble polymer was added was also prepared. And each performed nucleic acid amplification reaction by PCR.
  • each PCR reaction solution containing the nucleic acid amplification product obtained by PCR was dropped on the DNA chip as it was and allowed to stand at 45 ° C. for 1 hour for hybridization. That is, a PCR reaction solution to which neither a metal salt nor a water-soluble polymer is added, and a PCR reaction solution to which a metal salt is added and PEG600, PEG1000, PEG6000, PEG12000, PEG20000, PVP, Tween20, or Tween80 are added.
  • the hybridization was performed without mixing the hybridization buffer with the PCR reaction solution.
  • both the metal salt and the water-soluble polymer were used for the PCR reaction solution containing the nucleic acid amplification product obtained by adding and amplifying both the metal salt and the water-soluble polymer in the PCR reaction solution.
  • a PCR reaction solution mixed with a buffer without adding both a metal salt and a water-soluble polymer was used.
  • a fluorescence intensity comparable to that obtained is obtained. That is, it can be seen that in these PCR reaction solutions, hybridization was sufficiently carried out using the reaction solution containing the nucleic acid amplification product as it was by adding a metal salt and a water-soluble polymer.
  • the target region by the nucleic acid amplification reaction is only one region of vtx1, but other regions may be targeted, and multiple target regions are targeted and simultaneously amplified by the nucleic acid amplification reaction. It can also be applied to the case, and can be changed as appropriate.
  • the present invention can be suitably used for efficiently performing a nucleic acid amplification reaction by PCR or the like and a hybridization using a reaction solution containing the obtained nucleic acid amplification product.

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Abstract

L'invention concerne un procédé d'amplification et de détection d'acide nucléique, ledit procédé comprenant l'amplification d'une région cible dans un acide nucléique contenu dans un échantillon par une réaction d'amplification d'acide nucléique et puis la détection du produit d'amplification de l'acide nucléique par hybridation, le produit d'amplification de l'acide nucléique étant lié à un acide nucléique ayant une séquence de bases complémentaire de celui-ci; un sel métallique et/ou un polymère soluble dans l'eau étant ajoutés à une solution de réaction pour l'amplification d'acide nucléique et la réaction d'amplification d'acide nucléique étant effectuée à l'aide de la solution de réaction; et la solution de réaction contenant le produit d'amplification d'acide nucléique obtenu par la réaction d'amplification d'acide nucléique étant utilisée en tant que telle dans la réalisation de l'hybridation.
PCT/JP2017/029891 2016-08-24 2017-08-22 Procédé d'amplification et de détection d'acide nucléique, et solution d'amplification et de détection d'acide nucléique WO2018038088A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009086608A2 (fr) * 2007-12-26 2009-07-16 Eppendorf Array Technologies S.A. Procédé et kit pour effectuer une amplification par pcr et une détection de microréseau dans le même milieu et/ou la même chambre
WO2012049066A2 (fr) * 2010-10-12 2012-04-19 Eppendorf Ag Amplification en temps réel et détection basée sur un micro-réseau de cibles d'acide nucléique dans un test sur puce en continu
JP2015100332A (ja) * 2013-11-27 2015-06-04 東ソー株式会社 核酸の検出方法
JP2017169449A (ja) * 2016-03-18 2017-09-28 株式会社東芝 核酸検出方法

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Publication number Priority date Publication date Assignee Title
AT410218B (de) * 1999-08-20 2003-03-25 Baxter Ag Verfahren zur herstellung eines qualitätsgesicherten pools biologischer proben
JP6467829B2 (ja) * 2014-09-03 2019-02-13 東洋紡株式会社 改良されたｒｔ−ｐｃｒ反応

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009086608A2 (fr) * 2007-12-26 2009-07-16 Eppendorf Array Technologies S.A. Procédé et kit pour effectuer une amplification par pcr et une détection de microréseau dans le même milieu et/ou la même chambre
WO2012049066A2 (fr) * 2010-10-12 2012-04-19 Eppendorf Ag Amplification en temps réel et détection basée sur un micro-réseau de cibles d'acide nucléique dans un test sur puce en continu
JP2015100332A (ja) * 2013-11-27 2015-06-04 東ソー株式会社 核酸の検出方法
JP2017169449A (ja) * 2016-03-18 2017-09-28 株式会社東芝 核酸検出方法

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