WO2024069938A1 - Reaction vessel for nucleic acid amplification, cartridge, and nucleic acid detection method - Google Patents

Reaction vessel for nucleic acid amplification, cartridge, and nucleic acid detection method Download PDF

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Publication number
WO2024069938A1
WO2024069938A1 PCT/JP2022/036723 JP2022036723W WO2024069938A1 WO 2024069938 A1 WO2024069938 A1 WO 2024069938A1 JP 2022036723 W JP2022036723 W JP 2022036723W WO 2024069938 A1 WO2024069938 A1 WO 2024069938A1
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nucleic acid
reaction
reaction vessel
amplification
cartridge
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PCT/JP2022/036723
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French (fr)
Japanese (ja)
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健悟 臼井
ディミトリー プザンコフ
ニキータ オベチキン
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株式会社Mirai Genomics
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Priority to PCT/JP2022/036723 priority Critical patent/WO2024069938A1/en
Publication of WO2024069938A1 publication Critical patent/WO2024069938A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • 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

Definitions

  • the present invention relates to a reaction chamber for nucleic acid amplification, a cartridge, and a method for detecting nucleic acid.
  • Patent Document 1 discloses a small, portable amplification and detection device that makes it easier to amplify and detect nucleic acids.
  • Patent Document 1 International Publication No. WO 2018/123837, the entire disclosure of which is expressly incorporated herein by reference.
  • the device described in Patent Document 1 is a device that separately collects nucleic acid from a specimen, and then amplifies and detects the collected nucleic acid.
  • the inventors have come up with a plan to use the sample as is without pretreatment, and to recover nucleic acid from the sample, amplify the recovered nucleic acid, and detect the amplified nucleic acid in a single device.
  • the nucleic acid recovery, amplification, and detection processes will be carried out automatically and sequentially in chambers connected by a microchannel.
  • false positives occur due to the nucleic acid amplification reaction chamber in which the recovered nucleic acid is amplified.
  • the problem to be solved by the present invention is to provide a nucleic acid amplification reaction vessel capable of suppressing the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid detection method using this cartridge, and the present invention aims to provide a nucleic acid amplification reaction vessel capable of suppressing the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid detection method using this cartridge.
  • the present invention is as follows.
  • [1] A reaction vessel for nucleic acid amplification, the reaction vessel having an inner surface with a surface roughness Ra of 25 nm or less.
  • [2] The reaction vessel according to [1], wherein the reaction vessel is composed of a reaction vessel body and a film that shields an opening of the reaction vessel body, and the surface roughness Ra of the inner surface of the reaction vessel body and the film is 25 nm or less.
  • the reaction vessel body is an injection-molded product molded using an injection molding mold in which the surface for forming the inner surface of the reaction vessel body has a surface roughness Ra of 25 nm or less.
  • reaction vessel according to [1] or [2], wherein the reaction vessel is an injection-molded product having a wax coating on the inner surface of the reaction vessel, and the surface roughness Ra of the wax-coated inner surface is 25 nm or less.
  • the reaction vessel has a space for containing a reaction liquid, The reaction vessel according to any one of [1] to [4], wherein the reaction liquid-accommodating space has a volume in the range of 10 to 500 ⁇ L.
  • nucleic acid amplification reaction is an isothermal amplification reaction or a thermocycle amplification reaction of nucleic acid.
  • the reaction vessel according to [6], wherein the isothermal amplification reaction of nucleic acid is a nucleic acid LAMP method or a SmartAmp method.
  • a method for detecting a nucleic acid comprising using the cartridge according to [9], amplifying a target nucleic acid in a reaction chamber of the cartridge using a plurality of primers, and detecting the amplified nucleic acid.
  • the method for detecting a nucleic acid according to [11], wherein the isothermal amplification reaction of the nucleic acid is a LAMP method or a SmartAmp method of the nucleic acid.
  • the present invention makes it possible to suppress the occurrence of false positives in detection after nucleic acid amplification.
  • FIG. 1 shows a schematic diagram of an example of a reaction vessel of the present invention.
  • FIG. 2 shows the results of nucleic acid amplification reaction 1.
  • FIG. 3 shows the results of nucleic acid amplification reaction 2.
  • FIG. 4 shows the results of nucleic acid amplification reaction 3.
  • FIG. 5 shows the results of nucleic acid amplification reaction 4.
  • the present invention relates to a reaction chamber for amplifying nucleic acid, the inner surface of which has a surface roughness Ra of 25 nm or less.
  • the present invention by setting the surface roughness Ra of the inner surface of the reaction vessel to 25 nm or less, it is possible to suppress the amplification of nucleic acids other than the target nucleic acid, and to suppress the occurrence of false positives in detection after nucleic acid amplification.
  • FIG. 1 shows an example of a reaction vessel for nucleic acid amplification of the present invention. Below, the reaction vessel for nucleic acid amplification of the present invention will be described based on FIG. 1.
  • the reaction vessel 20 shown in FIG. 1 is composed of a reaction vessel body 10 and a film 40 that shields the opening of the reaction vessel space of the reaction vessel body 10.
  • the reaction vessel body 10 and the film 40 are shown separately, but the reaction vessel 20 is formed by providing the film 40 to shield the opening of the reaction vessel space of the reaction vessel body 10.
  • four reaction vessels 20 are formed.
  • the surface roughness Ra of the inner surface of the reaction vessel body 10 and the film is 25 nm or less, and the surface roughness Ra of the inner surface of the reaction vessel 20 is 25 nm or less.
  • the reaction tank body can be, for example, an injection molded product molded using an injection molding mold in which the surface for forming the inner surface of the reaction tank body has a surface roughness Ra of 25 nm or less.
  • An injection molded product molded using an injection molding mold in which the surface for molding has a surface roughness Ra of 25 nm or less will have a surface roughness Ra of 25 nm or less.
  • the material of the reaction tank body is not particularly limited, and general injection molding resins can be used.
  • the reaction tank body can be made of, for example, polypropylene.
  • the reaction tank body is, for example, an injection molded product having a wax coating on the inner surface of the reaction tank 20, and the surface roughness Ra of the wax-coated inner surface can be 25 nm or less. In this case, it is possible to obtain a reaction tank body having an inner surface with a surface roughness Ra of 25 nm or less without using an injection molding mold with a molding surface with a surface roughness Ra of 25 nm or less.
  • the film may be a resin film, for example, a polypropylene film, and may be a biaxially oriented film or a non-oriented film.
  • a biaxially oriented film is preferable, but in the present invention, there is no particular restriction as long as the surface roughness Ra is 25 nm or less.
  • a film 40 is provided to cover the opening of the reaction tank space of the reaction tank body 10, and by, for example, heat sealing, the opening of the reaction tank space is covered with the film 40, thereby obtaining an individual reaction tank 20.
  • the nucleic acid amplification reaction tank 20 of the present invention is, for example, a vertically elongated reaction tank, the lower part in the longitudinal direction being a reaction liquid storage space 21 and the upper part being a reaction liquid supply space 22, and the reaction liquid supply space 22 can have a reaction liquid supply port 32 and an exhaust port 24. Furthermore, at least a part near the boundary between the reaction liquid supply space 22 and the reaction liquid storage space 21 can have a protrusion 25 facing the inside of the reaction tank.
  • the present invention relates to a cartridge 10 for nucleic acid amplification, which has chambers connected by microchannels and includes one or more reaction chambers 20 for nucleic acid amplification connected to the chambers via microchannels 30 and 31, wherein the reaction chambers 20 are the reaction chambers of the present invention.
  • FIG. 1 shows an example of a cartridge having four reaction vessels 20.
  • the microchannel 30 from the upstream chamber branches into two along the way to form microchannels 31, which further branch into two to form a total of four channels, and these channels are connected to the reaction liquid supply ports 32 of each reaction vessel.
  • the supply of reaction liquid to the reaction vessel 20 through the microchannels 30 and 31 is performed by applying negative pressure to the reaction liquid supply space 22 from the exhaust port 24 located in the reaction liquid supply space 22. Negative pressure can be applied by connecting, for example, a vacuum pump to the exhaust port 24.
  • the vacuum pump can be provided separately from the cartridge.
  • the present invention relates to a method for detecting nucleic acid, which comprises using the cartridge of the present invention, amplifying a target nucleic acid in a reaction chamber of the cartridge using a plurality of primers, and detecting the amplified nucleic acid.
  • the nucleic acid amplification reaction can be an isothermal or thermocycling amplification reaction of nucleic acid.
  • the nucleic acid to be amplified is not particularly limited, and can be, for example, RNA or DNA.
  • the nucleic acid amplification reaction can be an isothermal amplification reaction or a thermocycling amplification reaction of nucleic acid.
  • the isothermal amplification reaction can be, for example, the LAMP method or the SmartAmp method.
  • the thermocycling amplification reaction can be a PCR amplification reaction.
  • the nucleic acid amplification enzyme is not particularly limited, but may be, for example, an enzyme for isothermal amplification reaction of nucleic acid or an enzyme for thermocycle amplification reaction.
  • the isothermal amplification reaction of nucleic acid may be, for example, the LAMP method or the SmartAmp method of nucleic acid, and may be an enzyme for amplification reaction of nucleic acid using a strand displacement reaction.
  • the polymerase which is an enzyme for nucleic acid amplification reactions having strand displacement activity, can be any known enzyme.
  • the polymerase described in WO 2004/040019 can be mentioned, but is not intended to be limited thereto.
  • the polymerase having strand displacement activity can also be DNA polymerase (Aac), which is disclosed in WO 2009/054510 (Japanese Patent No. 4450867).
  • any of those that are normal, mesophilic, or thermostable can be suitably used.
  • this polymerase may be either a natural form or a mutant with an artificial mutation.
  • Such a polymerase includes DNA polymerase.
  • DNA polymerase includes mutants of DNA polymerase derived from thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as "B.st”) and Bacillus caldotenax (hereinafter referred to as "B.ca”) that lack 5' ⁇ 3' exonuclease activity, and Klenow fragment of DNA polymerase I derived from Escherichia coli (E.
  • DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, ⁇ 29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9°Nm DNA polymerase, Therminator DNA polymerase, and Taq DNA polymerase.
  • RNA When the nucleic acid to be amplified is RNA, a reverse transcriptase can be used in addition to the DNA polymerase, or a DNA polymerase that also has reverse transcription activity can be used as the DNA polymerase.
  • the reverse transcriptase is not particularly limited as long as it has cDNA synthesis activity using RNA as a template, and examples of the reverse transcriptase include reverse transcriptases of various origins, such as avian myeloblastosis virus reverse transcriptase (AMVRTase), Rous-associated virus 2 reverse transcriptase (RAV-2RTase), and Moloney murine leukemia virus reverse transcriptase (MMLVRTase).
  • AMVRTase avian myeloblastosis virus reverse transcriptase
  • RAV-2RTase Rous-associated virus 2 reverse transcriptase
  • MMLVRTase Moloney murine leukemia virus reverse transcriptase
  • the primer is appropriately selected depending on the enzyme used for the nucleic acid amplification reaction.
  • the enzyme for the nucleic acid amplification reaction is an enzyme for the nucleic acid amplification reaction using a strand displacement reaction
  • examples of the primers include those described in International Publication No. 2004/040019, Japanese Patent Application Publication No. 2009-171935, Japanese Patent Application Publication No. 2011-50380, etc.
  • the method of the present invention may further include a step of optically detecting, electrically detecting, or detecting by surface plasmon resonance the nucleic acid amplified in the reaction vessel after the nucleic acid amplification operation.
  • the detection of the amplified nucleic acid may be performed using a fluorogenic primer as the primer and using the label of the fluorogenic primer.
  • the detection of the amplified nucleic acid may be performed by utilizing the exciton effect using an exciton primer or an exciton probe in the amplification reaction.
  • the method of optically detecting the nucleic acid may be a method using an intercalating dye.
  • the nucleic acid amplification reaction it is preferable to carry out the nucleic acid amplification reaction by the SmartAmp method or the LAMP method, and to detect the labeling with an exciton primer or an exciton probe.
  • nucleic acid melting curve can be drawn, and the melting curve can be used to determine the properties of the amplified product, such as whether it is a false positive or true positive.
  • reaction tank body was produced by injection molding polypropylene.
  • the surface of the injection mold was polished.
  • the surface roughness Ra at multiple points on the inner surface of the reaction tank body determined by AFM analysis was in the range of 9 to 13 nm.
  • a polypropylene film was heat-sealed to the opening of the reaction tank space of this reaction tank body to form a reaction tank.
  • This reaction tank is referred to as reaction tank 1.
  • the total volume of the reaction tank is 200 ⁇ L.
  • reaction chamber 2 Preparation of Reaction Chamber 2 Using an injection molding mold with an unpolished surface, polypropylene was injection molded to prepare the main body of a reaction chamber for nucleic acid amplification in the same manner as in reaction chamber 1. A polypropylene film similar to reaction chamber 1 was laser fused to the opening of the reaction chamber space of this reaction chamber main body to form a reaction chamber. This reaction chamber is designated reaction chamber 2.
  • reaction tank 3 A reaction tank body was prepared by injection molding polypropylene in the same manner as reaction tank 2. The total volume of the reaction tank was 200 ⁇ L. Next, the inner surface of the reaction tank body was wax-coated. The surface roughness Ra of multiple points on the wax-coated inner surface determined by AFM analysis was in the range of 9 to 13 nm. A polypropylene film similar to reaction tank 1 was heat-sealed to the opening of the reaction tank space of this reaction tank body to form a reaction tank. This reaction tank is designated reaction tank 3.
  • Nucleic acid amplification reactions 1 to 4 were carried out using the SmartAmp method. Nucleic acid amplification using the SmartAmp method was carried out under the following conditions. The reaction volume was 40 ⁇ L.
  • Folding primer (FP) Outer boost primer (oBP) Turn-back primer (TP), turn-back site Internal boost primer (iBP) Turn-back primer (TP), annealing site Outer primer (OP)
  • Amplification reaction buffer Trizma, pH 8.0 20 mM, CH3COOK 60 mM, ( NH4 ) 2SO4 20 mM, MgSO4 16 mM, Tween 20 0.2% (w/v), dNTP 1.4 mM, RNase free water)
  • Nucleic acid amplification reaction 1 Nucleic acid amplification (five times) was performed by the SmartAmp method using reaction vessel 1, using the same amount of water (negative control) and only the internal standard nucleic acid (internal positive control) instead of the target nucleic acid (RNA). The results are shown in Figure 2. Only the amplification of the internal standard nucleic acid was confirmed.
  • Nucleic acid amplification reaction 2 Nucleic acid amplification (three times) was performed by the SmartAmp method using only the target nucleic acid (RNA) and the internal standard nucleic acid in the reaction vessel 1. The results are shown in Figure 3. Amplification of the internal standard nucleic acid and the target nucleic acid (three lines indicated as positive control) were confirmed. However, Figure 3 also shows the results of nucleic acid amplification by the SmartAmp method using only the internal standard nucleic acid without using the target nucleic acid.
  • Nucleic acid amplification reaction 3 Nucleic acid amplification (six times) was carried out by the SmartAmp method using reaction vessel 2, using the same amount of water and only the internal standard nucleic acid instead of the target nucleic acid (RNA). The results are shown in Figure 4. In three of the six amplification reactions, not only the amplification of the internal standard nucleic acid but also the amplification of other nucleic acids was confirmed, suggesting the occurrence of false positives.
  • Nucleic acid amplification reaction 4 Nucleic acid amplification (four times) was carried out by the SmartAmp method using reaction vessel 3, using the same amount of water and only the internal standard nucleic acid instead of the target nucleic acid (RNA). The results are shown in Figure 5. Only the amplification of the internal standard nucleic acid was confirmed.
  • the surface roughness Ra of the inner surface of the reaction vessel is 25 nm or less, preferably in the range of 5 to 20 nm, more preferably in the range of 7 to 17 nm, and even more preferably in the range of 8 to 15 nm, which can suppress the amplification of nucleic acids other than the target nucleic acid, which is thought to be the cause of false positives.
  • the present invention is useful in fields related to nucleic acid amplification and detection.

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Abstract

The present invention pertains to a reaction vessel for nucleic acid amplification, wherein the surface roughness Ra of the inner surface of the reaction vessel is 25 nm or less, and to a cartridge (10) for nucleic acid amplification, said cartridge having chambers that communicate with each other via micro flow passages, and including one or more reaction vessels (20) for nucleic acid amplification that communicate with the chambers via micro flow passages (30 and 31), wherein each of the reaction vessels (20) is the aforementioned reaction vessel. In addition, the present invention pertains to a nucleic acid detection method wherein a plurality of primers are used in the reaction vessel of the cartridge to amplify a target nucleic acid, and the amplified nucleic acid is detected. The present invention provides a reaction vessel for nucleic acid amplification with which the occurrence of false positives can be suppressed, a cartridge for nucleic acid amplification including said vessel, and a nucleic acid detection method.

Description

核酸増幅用反応槽、カートリッジ及び核酸検出方法Reaction chamber for amplifying nucleic acid, cartridge and method for detecting nucleic acid
　本発明は、核酸増幅用反応槽、カートリッジ及び核酸検出方法に関する。 The present invention relates to a reaction chamber for nucleic acid amplification, a cartridge, and a method for detecting nucleic acid.
　ＣＯＶＩＤ－１９の蔓延に伴って、ウイルス中の核酸を回収し、増幅し、検出して、ウイルス感染の有無を簡便にかつ確実に判定する技術に対する要求は日々高まっている。また、ＣＯＶＩＤ－１９の問題を解消した後にも、人類は様々なウイルスや細菌による疾病に直面していることから、同様の技術に対する要求は依然として存在する。 As COVID-19 spreads, there is a growing demand for technology that can extract, amplify, and detect nucleic acids in the virus to easily and reliably determine whether or not a person is infected with the virus. Even after the COVID-19 problem is resolved, there will still be a demand for similar technology, as humanity will be faced with a variety of viral and bacterial diseases.
　核酸を増幅し、検出する技術は、サーモサイクラーを用いたＰＣＲ法の出現と、その後のサーモサイクルＰＣＲ法以外の様々な方法の発展により、方法論としてはほぼ確立され、実験室のみならず、病院の検査室などにおいても簡便に行われるようになってきた。しかし、それでも、ウイルス等の生体試料からの核酸回収をマニュアル操作で行った後に、核酸の増幅と検出を既存の増幅検出装置を用いて行うことが多かった。しかし、ＣＯＶＩＤ－１９の蔓延に伴って、既存の方法及び装置を用いて、保健所や検査会社に検体を持ち込んでの対応では深刻な自体に対応できないことは明らかである。多量の検体を、場所を選ばず短時間にかつ簡易に処理して、ウイルス感染の有無を、確実に判定することはできないのが現状である。 The technology for amplifying and detecting nucleic acids has become almost established as a methodology with the advent of PCR using a thermocycler and the subsequent development of various methods other than thermocycle PCR, and it has become easy to perform not only in laboratories but also in hospital examination rooms. However, even so, it was often the case that nucleic acid was manually extracted from biological samples such as viruses, and then the nucleic acid was amplified and detected using existing amplification and detection devices. However, with the spread of COVID-19, it is clear that the serious situation cannot be dealt with by using existing methods and devices and bringing samples to public health centers or testing companies. The current situation is that it is not possible to reliably determine the presence or absence of viral infection by processing a large number of samples quickly and easily, regardless of location.
　特許文献１には、核酸の増幅と検出をより簡易に行える、携帯可能な小型の増幅検出装置が開示されている。 Patent Document 1 discloses a small, portable amplification and detection device that makes it easier to amplify and detect nucleic acids.
特許文献１：国際公開第２０１８／１２３８３７号
特許文献１の全記載は、ここに特に開示として援用される。 Patent Document 1: International Publication No. WO 2018/123837, the entire disclosure of which is expressly incorporated herein by reference.
　特許文献１に記載の装置では、検体から核酸を別途回収した後、回収した核酸を増幅及び検出する装置である。しかし、この装置を用いる方法では、検体から核酸を別途回収する必要があり、検体から一度にウイルスの検出はできない。 The device described in Patent Document 1 is a device that separately collects nucleic acid from a specimen, and then amplifies and detects the collected nucleic acid. However, with the method using this device, it is necessary to separately collect nucleic acid from the specimen, and it is not possible to detect viruses from the specimen all at once.
　これに対し、本発明者らは、検体を前処理することなくそのまま用いて、検体からの核酸の回収、回収した核酸の増幅、及び増幅した核酸の検出を１つの装置で行うことを企画した。この装置では、上記核酸の回収、増幅、検出の工程を、マイクロ流路で連絡したチャンバーにおいて逐次自動で実施することを想定する。しかし、その中で、回収した核酸の増幅を行う核酸増幅用反応槽に起因して偽陽性が生じることを突き止めた。 In response to this, the inventors have come up with a plan to use the sample as is without pretreatment, and to recover nucleic acid from the sample, amplify the recovered nucleic acid, and detect the amplified nucleic acid in a single device. In this device, it is envisioned that the nucleic acid recovery, amplification, and detection processes will be carried out automatically and sequentially in chambers connected by a microchannel. However, they have discovered that false positives occur due to the nucleic acid amplification reaction chamber in which the recovered nucleic acid is amplified.
　本発明が解決すべき課題は、偽陽性の発生を抑制できる核酸増幅用反応槽、それを含む核酸増幅用カートリッジ、及びこのカートリッジを用いる核酸検出方法を提供することにあり、本発明は、偽陽性の発生を抑制できる核酸増幅用反応槽、それを含む核酸増幅用カートリッジ、及びこのカートリッジを用いる核酸検出方法を提供することを目的とする。 The problem to be solved by the present invention is to provide a nucleic acid amplification reaction vessel capable of suppressing the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid detection method using this cartridge, and the present invention aims to provide a nucleic acid amplification reaction vessel capable of suppressing the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid detection method using this cartridge.
　本発明者らの検討の結果、偽陽性発生の原因となり得る、目的核酸以外の核酸増幅は、射出成形で作製した反応槽の内面の表面が粗く、その結果、核酸増幅反応においてプライマーがダイマ等を生成し、それが増幅されることが原因であると判明した。このことは、核酸増幅反応をＳｍａｒｔＡｍｐ法で行った場合に顕著であった。さらに、反応槽の内表面の表面粗さＲａを２５μｍ以下にすることで、目的核酸以外の核酸増幅を抑制できることを見いだして本発明を完成させた。 As a result of the inventors' investigations, it was found that the amplification of nucleic acids other than the target nucleic acid, which can cause false positives, is caused by the rough inner surface of the reaction vessel produced by injection molding, which results in the primers generating dimers and the like during the nucleic acid amplification reaction, which are then amplified. This was particularly evident when the nucleic acid amplification reaction was carried out using the SmartAmp method. Furthermore, it was discovered that the amplification of nucleic acids other than the target nucleic acid can be suppressed by setting the surface roughness Ra of the inner surface of the reaction vessel to 25 μm or less, which led to the completion of the present invention.
　本発明は、以下の通りである。
[１]
　核酸増幅用反応槽であって、反応槽の内表面の表面粗さＲａが２５ｎｍ以下である、前記反応槽。
[２]
　前記反応槽は、反応槽本体と反応槽本体の開口を遮蔽するフィルムとで構成され、反応槽本体の内表面及びフィルムの表面粗さＲａが２５ｎｍ以下である、［１］に記載の反応槽。
［３］
　反応槽本体は、反応槽本体の内表面形成用表面の表面粗さＲａが２５ｎｍ以下である射出成形用型を用いて成形された射出成形品である、［１］または［２］に記載の反応槽。
［４］
　前記反応槽は、反応槽の内表面にワックス被覆を有する射出成形品であり、ワックス被覆した内表面の表面粗さＲａが２５ｎｍ以下である、［１］または［２］に記載の反応槽。
［５］
　前記反応槽は、反応液収容用空間を有し、
　前記反応液収容用空間は、１０～５００μＬの範囲の容積を有する、［１］～［４］のいずれか１項に記載の反応槽。
［６］
　核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応である、［１］～［５］のいずれか１項に記載の反応槽。
［７］
　核酸の等温増幅反応は、核酸のＬＡＭＰ法またはＳｍａｒｔＡｍｐ法である、[６]に記載の反応槽。
［８］
　核酸の等温増幅反応を、エキシトンプライマーまたはエキシトンプローブで標識検出する、［７］に記載の反応槽。
［９］
　マイクロ流路で連絡されたチャンバーを有し、マイクロ流路（３０及び３１）を介して前記チャンバーと連絡する１つまたは２つ以上の核酸増幅用反応槽（２０）を含む核酸増幅用カートリッジ（１０）であって、
　前記反応槽（２０）が、［１］～［８］のいずれか１項に記載の反応槽である、前記カートリッジ。
［１０］
　［９］に記載のカートリッジを用い、前記カートリッジの反応槽中で、複数のプライマーを用いて目的核酸を増幅し、増幅した核酸を検出する、核酸の検出方法。
［１１］
　核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応である、［１０］に記載の核酸の検出方法。
［１２］
　核酸の等温増幅反応は、核酸のＬＡＭＰ法またはＳｍａｒｔＡｍｐ法である、［１１］に記載の核酸の検出方法。
［１３］
　核酸の等温増幅反応を、エキシトンプライマーまたはエキシトンプローブで標識検出する、［１２］に記載の核酸の検出方法。 The present invention is as follows.
[1]
A reaction vessel for nucleic acid amplification, the reaction vessel having an inner surface with a surface roughness Ra of 25 nm or less.
[2]
The reaction vessel according to [1], wherein the reaction vessel is composed of a reaction vessel body and a film that shields an opening of the reaction vessel body, and the surface roughness Ra of the inner surface of the reaction vessel body and the film is 25 nm or less.
[3]
The reaction vessel according to [1] or [2], wherein the reaction vessel body is an injection-molded product molded using an injection molding mold in which the surface for forming the inner surface of the reaction vessel body has a surface roughness Ra of 25 nm or less.
[4]
The reaction vessel according to [1] or [2], wherein the reaction vessel is an injection-molded product having a wax coating on the inner surface of the reaction vessel, and the surface roughness Ra of the wax-coated inner surface is 25 nm or less.
[5]
The reaction vessel has a space for containing a reaction liquid,
The reaction vessel according to any one of [1] to [4], wherein the reaction liquid-accommodating space has a volume in the range of 10 to 500 μL.
[6]
The reaction vessel according to any one of [1] to [5], wherein the nucleic acid amplification reaction is an isothermal amplification reaction or a thermocycle amplification reaction of nucleic acid.
[7]
The reaction vessel according to [6], wherein the isothermal amplification reaction of nucleic acid is a nucleic acid LAMP method or a SmartAmp method.
[8]
The reaction vessel according to [7], in which an isothermal amplification reaction of nucleic acid is detected by labeling with an exciton primer or an exciton probe.
[9]
A nucleic acid amplification cartridge (10) having chambers connected by a microchannel, the cartridge including one or more nucleic acid amplification reaction chambers (20) connected to the chambers via microchannels (30 and 31),
The cartridge, wherein the reaction vessel (20) is the reaction vessel described in any one of [1] to [8].
[10]
A method for detecting a nucleic acid, comprising using the cartridge according to [9], amplifying a target nucleic acid in a reaction chamber of the cartridge using a plurality of primers, and detecting the amplified nucleic acid.
[11]
The method for detecting a nucleic acid according to [10], wherein the nucleic acid amplification reaction is an isothermal amplification reaction or a thermocycle amplification reaction of a nucleic acid.
[12]
The method for detecting a nucleic acid according to [11], wherein the isothermal amplification reaction of the nucleic acid is a LAMP method or a SmartAmp method of the nucleic acid.
[13]
The method for detecting a nucleic acid according to [12], wherein an isothermal amplification reaction of a nucleic acid is detected by labeling with an exciton primer or an exciton probe.
　本発明によれば、核酸増幅した後の検出において偽陽性の発生を抑制することができる。 The present invention makes it possible to suppress the occurrence of false positives in detection after nucleic acid amplification.
図１は、本発明の反応槽の一例の概略図を示す。FIG. 1 shows a schematic diagram of an example of a reaction vessel of the present invention. 図２は、核酸増幅反応１の結果を示す。FIG. 2 shows the results of nucleic acid amplification reaction 1. 図３は、核酸増幅反応２の結果を示す。FIG. 3 shows the results of nucleic acid amplification reaction 2. 図４は、核酸増幅反応３の結果を示す。FIG. 4 shows the results of nucleic acid amplification reaction 3. 図５は、核酸増幅反応４の結果を示す。FIG. 5 shows the results of nucleic acid amplification reaction 4.
＜核酸増幅用反応槽＞
　本発明は、反応槽の内表面の表面粗さＲａが２５ｎｍ以下である、核酸増幅用反応槽に関する。 <Nucleic acid amplification reaction chamber>
The present invention relates to a reaction chamber for amplifying nucleic acid, the inner surface of which has a surface roughness Ra of 25 nm or less.
　本発明においては、反応槽の内表面の表面粗さＲａが２５ｎｍ以下であることで、目的核酸以外の核酸増幅を抑制でき、核酸増幅した後の検出において偽陽性の発生を抑制することができる。 In the present invention, by setting the surface roughness Ra of the inner surface of the reaction vessel to 25 nm or less, it is possible to suppress the amplification of nucleic acids other than the target nucleic acid, and to suppress the occurrence of false positives in detection after nucleic acid amplification.
　図１は、本発明の核酸増幅用反応槽の一例を示すものであり、以下、図１に基づいて、本発明の核酸増幅用反応槽について説明する。 FIG. 1 shows an example of a reaction vessel for nucleic acid amplification of the present invention. Below, the reaction vessel for nucleic acid amplification of the present invention will be described based on FIG. 1.
　図１に示す反応槽２０は、反応槽本体１０と、反応槽本体１０の反応槽空間の開口を遮蔽するフィルム４０とで構成される。図１では、反応槽本体１０とフィルム４０は別々に記載されているが、反応槽本体１０の反応槽空間の開口を遮蔽するようにフィルム４０が設けられることにより、反応槽２０が形成される。図１では４つの反応槽２０が形成される。反応槽本体１０の内表面及びフィルム（少なくとも反応槽空間側の表面）の表面粗さＲａは、２５ｎｍ以下であり、反応槽２０の内表面の表面粗さＲａは、２５ｎｍ以下となる。 The reaction vessel 20 shown in FIG. 1 is composed of a reaction vessel body 10 and a film 40 that shields the opening of the reaction vessel space of the reaction vessel body 10. In FIG. 1, the reaction vessel body 10 and the film 40 are shown separately, but the reaction vessel 20 is formed by providing the film 40 to shield the opening of the reaction vessel space of the reaction vessel body 10. In FIG. 1, four reaction vessels 20 are formed. The surface roughness Ra of the inner surface of the reaction vessel body 10 and the film (at least the surface on the reaction vessel space side) is 25 nm or less, and the surface roughness Ra of the inner surface of the reaction vessel 20 is 25 nm or less.
　反応槽本体は、例えば、反応槽本体の内表面形成用表面の表面粗さＲａが２５ｎｍ以下である射出成形用型を用いて成形された射出成形品であることができる。成形用表面の表面粗さＲａが２５ｎｍ以下である射出成形用型を用いて成形された射出成形品の表面粗さＲａが２５ｎｍ以下となる。反応槽本体の材料は、特に制限されず、一般の射出成形用樹脂を使用することができる。反応槽本体は、例えば、ポリプロピレン製であることができる。 The reaction tank body can be, for example, an injection molded product molded using an injection molding mold in which the surface for forming the inner surface of the reaction tank body has a surface roughness Ra of 25 nm or less. An injection molded product molded using an injection molding mold in which the surface for molding has a surface roughness Ra of 25 nm or less will have a surface roughness Ra of 25 nm or less. The material of the reaction tank body is not particularly limited, and general injection molding resins can be used. The reaction tank body can be made of, for example, polypropylene.
　反応槽本体は、例えば、反応槽２０の内表面にワックス被覆を有する射出成形品であり、ワックス被覆した内表面の表面粗さＲａが２５ｎｍ以下であることもできる。この場合は、射出成形に、成形用表面の表面粗さＲａが２５ｎｍ以下である射出成形用型を用いずとも、内表面の表面粗さＲａが２５ｎｍ以下手ある反応槽本体を得ることができる。 The reaction tank body is, for example, an injection molded product having a wax coating on the inner surface of the reaction tank 20, and the surface roughness Ra of the wax-coated inner surface can be 25 nm or less. In this case, it is possible to obtain a reaction tank body having an inner surface with a surface roughness Ra of 25 nm or less without using an injection molding mold with a molding surface with a surface roughness Ra of 25 nm or less.
　フィルムは、樹脂製のフィルムであることができ、例えば、ポリプロピレン製フィルムであることができ、二軸延伸フィルムまたは無延伸フィルムであることができる。強度及び透明性を考慮すると二軸延伸フィルムであることが好ましいが、本発明においては表面粗さＲａが２５ｎｍ以下であれば、特に制限はない。 The film may be a resin film, for example, a polypropylene film, and may be a biaxially oriented film or a non-oriented film. In consideration of strength and transparency, a biaxially oriented film is preferable, but in the present invention, there is no particular restriction as long as the surface roughness Ra is 25 nm or less.
　反応槽本体１０の反応槽空間の開口を遮蔽するようにフィルム４０を設け、例えば、熱融着することにより、反応槽空間の開口がフィルム４０で被覆されて個別の反応槽２０を得ることができる。 A film 40 is provided to cover the opening of the reaction tank space of the reaction tank body 10, and by, for example, heat sealing, the opening of the reaction tank space is covered with the film 40, thereby obtaining an individual reaction tank 20.
　本発明の核酸増幅用反応槽２０は、例えば、縦長形状の反応槽であって、長手方向の下部が反応液収容用空間２１であり、上部が反応液供給用空間２２であり、反応液供給用空間２２に反応液供給口３２及び排気口２４を有することができる。さらに、反応液供給用空間２２と反応液収容用空間２１の境界近傍の少なくとも一部に、反応槽内部に向かう突起部２５を有することもできる。 The nucleic acid amplification reaction tank 20 of the present invention is, for example, a vertically elongated reaction tank, the lower part in the longitudinal direction being a reaction liquid storage space 21 and the upper part being a reaction liquid supply space 22, and the reaction liquid supply space 22 can have a reaction liquid supply port 32 and an exhaust port 24. Furthermore, at least a part near the boundary between the reaction liquid supply space 22 and the reaction liquid storage space 21 can have a protrusion 25 facing the inside of the reaction tank.
＜核酸増幅用カートリッジ＞
　本発明は、マイクロ流路で連絡されたチャンバーを有し、マイクロ流路３０及び３１を介して上記チャンバーと連絡する１つまたは２つ以上の核酸増幅用反応槽２０を含む核酸増幅用カートリッジ１０であって、反応槽２０が、上記本発明の反応槽であるカートリッジに関する。 <Nucleic acid amplification cartridge>
The present invention relates to a cartridge 10 for nucleic acid amplification, which has chambers connected by microchannels and includes one or more reaction chambers 20 for nucleic acid amplification connected to the chambers via microchannels 30 and 31, wherein the reaction chambers 20 are the reaction chambers of the present invention.
　マイクロ流路で連絡されたチャンバーとマイクロ流路及び各マイクロ流路の連絡方法や構造には特に制限はない。本発明のカートリッジが有する反応槽２０の数は、１つまたは２つ以上であることができ、２つ以上は、３、４、５、６、７、８、９又は１０であることができるが、これらの数に限定されるものではない。図１には、４つの反応槽２０を有するカートリッジの例を示す。上流のチャンバーからのマイクロ流路３０が途中で２つに枝分かれしてマイクロ流路３１となり、マイクロ流路３１がさらに２つに枝分かれして全部で４つの流路を形成し、これらの流路が、反応槽のそれぞれの反応液供給口３２に連絡している。マイクロ流路３０、３１を介しての反応液の反応槽２０への供給は、反応液供給用空間２２内に位置する排気口２４から反応液供給用空間２２内に陰圧が付与されることにより、行われる。排気口２４に、例えば、減圧ポンプを接続することで、陰圧は付与できる。減圧ポンプは、カートリッジとは別に設けることができる。 There are no particular limitations on the chambers and microchannels connected by the microchannels, and on the method and structure of connection between each microchannel. The number of reaction vessels 20 in the cartridge of the present invention can be one or two or more, and two or more can be three, four, five, six, seven, eight, nine, or ten, but is not limited to these numbers. FIG. 1 shows an example of a cartridge having four reaction vessels 20. The microchannel 30 from the upstream chamber branches into two along the way to form microchannels 31, which further branch into two to form a total of four channels, and these channels are connected to the reaction liquid supply ports 32 of each reaction vessel. The supply of reaction liquid to the reaction vessel 20 through the microchannels 30 and 31 is performed by applying negative pressure to the reaction liquid supply space 22 from the exhaust port 24 located in the reaction liquid supply space 22. Negative pressure can be applied by connecting, for example, a vacuum pump to the exhaust port 24. The vacuum pump can be provided separately from the cartridge.
＜核酸の検出方法＞
　本発明は、本発明のカートリッジを用い、カートリッジの反応槽中で、複数のプライマーを用いて目的核酸を増幅し、増幅した核酸を検出する、核酸の検出方法に関する。 <Method for detecting nucleic acid>
The present invention relates to a method for detecting nucleic acid, which comprises using the cartridge of the present invention, amplifying a target nucleic acid in a reaction chamber of the cartridge using a plurality of primers, and detecting the amplified nucleic acid.
　核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応であることができる。 The nucleic acid amplification reaction can be an isothermal or thermocycling amplification reaction of nucleic acid.
　増幅されるべき核酸は、特に制限はないが、例えば、ＲＮＡまたはＤＮＡであることができる。核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応であることができる。等温増幅反応は、例えば、ＬＡＭＰ法またはＳｍａｒｔＡｍｐ法である。サーモサイクル増幅反応は、ＰＣＲ増幅反応であることができる。 The nucleic acid to be amplified is not particularly limited, and can be, for example, RNA or DNA. The nucleic acid amplification reaction can be an isothermal amplification reaction or a thermocycling amplification reaction of nucleic acid. The isothermal amplification reaction can be, for example, the LAMP method or the SmartAmp method. The thermocycling amplification reaction can be a PCR amplification reaction.
　核酸増幅用酵素は、特に制限されないが、例えば、核酸の等温増幅反応用酵素またはサーモサイクル増幅反応用酵素であることができる。核酸の等温増幅反応は、例えば、核酸のＬＡＭＰ法またはＳｍａｒｔＡｍｐ法であることができ、鎖置換反応を利用した核酸の増幅反応用酵素であることができる。 The nucleic acid amplification enzyme is not particularly limited, but may be, for example, an enzyme for isothermal amplification reaction of nucleic acid or an enzyme for thermocycle amplification reaction. The isothermal amplification reaction of nucleic acid may be, for example, the LAMP method or the SmartAmp method of nucleic acid, and may be an enzyme for amplification reaction of nucleic acid using a strand displacement reaction.
　鎖置換活性を有する核酸増幅反応用酵素であるポリメラーゼは、公知の酵素を利用できる。例えば、国際公開第２００４／０４００１９号に記載のポリメラーゼを挙げることができるが、これに限定される意図ではない。鎖置換活性を有するポリメラーゼは、ＤＮＡポリメラーゼ（Ａａｃ）であることもでき、国際公開第２００９／０５４５１０号（日本特許第４４５０８６７号）に開示されている。 The polymerase, which is an enzyme for nucleic acid amplification reactions having strand displacement activity, can be any known enzyme. For example, the polymerase described in WO 2004/040019 can be mentioned, but is not intended to be limited thereto. The polymerase having strand displacement activity can also be DNA polymerase (Aac), which is disclosed in WO 2009/054510 (Japanese Patent No. 4450867).
　鎖置換活性を有する核酸増幅反応に用いられるポリメラーゼとしては、常温性、中温性、もしくは耐熱性のいずれのものも好適に使用できる。また、このポリメラーゼは、天然体もしくは人工的に変異を加えた変異体のいずれであってもよい。このようなポリメラーゼとしては、ＤＮＡポリメラーゼが挙げられる。このようなＤＮＡポリメラーゼとしては、バチルス・ステアロサーモフィルス（Ｂａｃｉｌｌｕｓ　ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ、以下「Ｂ．ｓｔ」という）、バチルス・カルドテナックス（Ｂａｃｉｌｌｕｓ　ｃａｌｄｏｔｅｎａｘ、以下「Ｂ．ｃａ」という）等の好熱性バチルス属細菌由来ＤＮＡポリメラーゼの５’→３’エキソヌクレアーゼ活性を欠失した変異体、大腸菌（Ｅ．ｃｏｌｉ）由来ＤＮＡポリメラーゼＩのクレノウフラグメント等が挙げられる。核酸増幅反応において使用するＤＮＡポリメラーゼとしては、さらに、Ｖｅｎｔ　ＤＮＡポリメラーゼ、Ｖｅｎｔ（Ｅｘｏ－）ＤＮＡポリメラーゼ、ＤｅｅｐＶｅｎｔ　ＤＮＡポリメラーゼ、ＤｅｅｐＶｅｎｔ（Ｅｘｏ－）ＤＮＡポリメラーゼ、Φ２９ファージＤＮＡポリメラーゼ、ＭＳ－２ファージＤＮＡポリメラーゼ、Ｚ－Ｔａｑ　ＤＮＡポリメラーゼ、Ｐｆｕ　ＤＮＡポリメラーゼ、Ｐｆｕ　ｔｕｒｂｏ　ＤＮＡポリメラーゼ、ＫＯＤ　ＤＮＡポリメラーゼ、９°Ｎｍ　ＤＮＡポリメラーゼ、Ｔｈｅｒｍｉｎａｔｏｒ　ＤＮＡポリメラーゼ、Ｔａｑ　ＤＮＡポリメラーゼ等が挙げられる。 As a polymerase used in a nucleic acid amplification reaction having strand displacement activity, any of those that are normal, mesophilic, or thermostable can be suitably used. In addition, this polymerase may be either a natural form or a mutant with an artificial mutation. Such a polymerase includes DNA polymerase. Such DNA polymerase includes mutants of DNA polymerase derived from thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as "B.st") and Bacillus caldotenax (hereinafter referred to as "B.ca") that lack 5'→3' exonuclease activity, and Klenow fragment of DNA polymerase I derived from Escherichia coli (E. coli). Further examples of DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9°Nm DNA polymerase, Therminator DNA polymerase, and Taq DNA polymerase.
　増幅されるべき核酸がＲＮＡである場合は、ＤＮＡポリメラーゼに加えて逆転写酵素を併用するか、またはＤＮＡポリメラーゼとして、逆転写活性を併せ持つＤＮＡポリメラーゼを用いることもできる。逆転写酵素は、ＲＮＡを鋳型としたｃＤＮＡ合成活性を有するものであれば特に限定されず、例えば、トリ骨髄芽球症ウイルス由来逆転写酵素（ＡＭＶＲＴａｓｅ）、ラウス関連ウイルス２逆転写酵素（ＲＡＶ－２ＲＴａｓｅ）、モロニーネズミ白血病ウイルス由来逆転写酵素（ＭＭＬＶ　ＲＴａｓｅ）等、種々の起源の逆転写酵素が挙げられる。逆転写活性を併せ持つＤＮＡポリメラーゼとして、例えば、ＢｃａＢＥＳＴ　ＤＮＡポリメラーゼ、Ｂｃａ（ｅｘｏ－）ＤＮＡポリメラーゼ、Ｔｔｈ　ＤＮＡポリメラーゼ等を挙げることができる。 When the nucleic acid to be amplified is RNA, a reverse transcriptase can be used in addition to the DNA polymerase, or a DNA polymerase that also has reverse transcription activity can be used as the DNA polymerase. The reverse transcriptase is not particularly limited as long as it has cDNA synthesis activity using RNA as a template, and examples of the reverse transcriptase include reverse transcriptases of various origins, such as avian myeloblastosis virus reverse transcriptase (AMVRTase), Rous-associated virus 2 reverse transcriptase (RAV-2RTase), and Moloney murine leukemia virus reverse transcriptase (MMLVRTase). Examples of DNA polymerases that also have reverse transcription activity include BcaBEST DNA polymerase, Bca(exo-)DNA polymerase, and Tth DNA polymerase.
　プライマーは、核酸の増幅反応用酵素に応じて適宜選択される。核酸の増幅反応用酵素が、鎖置換反応を利用した核酸の増幅反応用酵素である場合は、国際公開第２００４／０４００１９号、特開２００９－１７１９３５号公報、特開２０１１－５０３８０号公報などに記載のプライマーを挙げることができる。 The primer is appropriately selected depending on the enzyme used for the nucleic acid amplification reaction. When the enzyme for the nucleic acid amplification reaction is an enzyme for the nucleic acid amplification reaction using a strand displacement reaction, examples of the primers include those described in International Publication No. 2004/040019, Japanese Patent Application Publication No. 2009-171935, Japanese Patent Application Publication No. 2011-50380, etc.
　本発明の方法は、核酸増幅操作後に、反応槽内で増幅された核酸を、光学的に検出する工程、電気的に検出する工程、または表面プラズモン共鳴により検出する工程をさらに含むことができる。増幅された核酸の検出は、プライマーとしてフルオロジェニックプライマーを用い、フルオロジェニックプライマーの標識を用いておこなうことができる。増幅された核酸の検出は、増幅反応においてエキシトンプライマーまたはエキシトンプローブを用いて、エキシトン効果を利用して行うことができる。核酸を光学的に検出する方法は、インターカレート色素を利用した方法でもよい。 The method of the present invention may further include a step of optically detecting, electrically detecting, or detecting by surface plasmon resonance the nucleic acid amplified in the reaction vessel after the nucleic acid amplification operation. The detection of the amplified nucleic acid may be performed using a fluorogenic primer as the primer and using the label of the fluorogenic primer. The detection of the amplified nucleic acid may be performed by utilizing the exciton effect using an exciton primer or an exciton probe in the amplification reaction. The method of optically detecting the nucleic acid may be a method using an intercalating dye.
　本発明の方法において、核酸増幅反応をＳｍａｒｔＡｍｐ法またはＬＡＭＰ法で行い、エキシトンプライマーまたはエキシトンプローブで標識検出することが好ましい。或いは、核酸増幅反応をＰＣＲ法で行い、エキシトンプライマーまたはエキシトンプローブで標識検出することが好ましい。 In the method of the present invention, it is preferable to carry out the nucleic acid amplification reaction by the SmartAmp method or the LAMP method, and to detect the labeling with an exciton primer or an exciton probe. Alternatively, it is preferable to carry out the nucleic acid amplification reaction by the PCR method, and to detect the labeling with an exciton primer or an exciton probe.
　核酸増幅反応に引き続き、核酸融解曲線を描き、融解曲線により、偽陽性や真の陽性の判定など、増幅産物の性質について判定することもできる。 Following the nucleic acid amplification reaction, a nucleic acid melting curve can be drawn, and the melting curve can be used to determine the properties of the amplified product, such as whether it is a false positive or true positive.
　以下、本発明を実施例に基づいて更に詳細に説明する。但し、実施例は本発明の例示であって、本発明は実施例に限定される意図ではない。 The present invention will be described in more detail below based on examples. However, the examples are merely illustrative of the present invention, and the present invention is not intended to be limited to the examples.
反応槽１の調製
　ポリプロピレンを射出成形して反応槽本体を作製した。射出成形用成形型の表面はポリッシングされたものである。ＡＦＭ分析により決定した反応槽本体の内表面における複数箇所の表面粗さＲａは、９～１３ｎｍの範囲であった。この反応槽本体の反応槽空間の開口部にポリプロピレン製フィルムを熱融着して反応槽を形成した。この反応槽を反応槽１とする。反応槽の全体の容量は２００μＬである。 Preparation of Reaction Tank 1 A reaction tank body was produced by injection molding polypropylene. The surface of the injection mold was polished. The surface roughness Ra at multiple points on the inner surface of the reaction tank body determined by AFM analysis was in the range of 9 to 13 nm. A polypropylene film was heat-sealed to the opening of the reaction tank space of this reaction tank body to form a reaction tank. This reaction tank is referred to as reaction tank 1. The total volume of the reaction tank is 200 μL.
反応槽２の調製
　表面をポリッシングしていない射出成形用成形型を用いて、反応槽１と同様にポリプロピレンを射出成形して核酸増幅用反応槽の本体を作製した。この反応槽本体の反応槽空間の開口部に反応槽１と同様のポリプロピレン製フィルムをレーザー融着して反応槽を形成した。この反応槽を反応槽２とする。 Preparation of Reaction Chamber 2 Using an injection molding mold with an unpolished surface, polypropylene was injection molded to prepare the main body of a reaction chamber for nucleic acid amplification in the same manner as in reaction chamber 1. A polypropylene film similar to reaction chamber 1 was laser fused to the opening of the reaction chamber space of this reaction chamber main body to form a reaction chamber. This reaction chamber is designated reaction chamber 2.
反応槽３の調製
　反応槽２と同様にポリプロピレンを射出成形して反応槽本体を作製した。反応槽の全体の容量は２００μＬである。次に、反応槽本体の内表面を、ワックス被覆した。ＡＦＭ分析により決定したワックス被覆内表面の複数箇所の表面粗さＲａは、９～１３ｎｍの範囲であった。この反応槽本体の反応槽空間の開口部に反応槽１と同様のポリプロピレン製フィルムを熱融着して反応槽を形成した。この反応槽を反応槽３とする。 Preparation of Reaction Tank 3 A reaction tank body was prepared by injection molding polypropylene in the same manner as reaction tank 2. The total volume of the reaction tank was 200 μL. Next, the inner surface of the reaction tank body was wax-coated. The surface roughness Ra of multiple points on the wax-coated inner surface determined by AFM analysis was in the range of 9 to 13 nm. A polypropylene film similar to reaction tank 1 was heat-sealed to the opening of the reaction tank space of this reaction tank body to form a reaction tank. This reaction tank is designated reaction tank 3.
　ＳｍａｒｔＡｍｐ法による核酸増幅により核酸増幅反応１～４を実施した。ＳｍａｒｔＡｍｐ法による核酸増幅は、以下の条件で実施した。反応容量は４０μＬとした。 Nucleic acid amplification reactions 1 to 4 were carried out using the SmartAmp method. Nucleic acid amplification using the SmartAmp method was carried out under the following conditions. The reaction volume was 40 μL.
酵素
ＤＮＡポリメラーゼ（Ａａｃ）　３４　Ｕ／ｒｅａｃｔｉｏｎ、
転写酵素（ＡＭＶ－ＲＴ）　２Ｕ／ｒｅａｃｔｉｏｎ Enzyme DNA polymerase (Aac) 34 U/reaction,
Transcriptase (AMV-RT) 2U/reaction
プライマー
Ｆｏｌｄｉｎｇ　ｐｒｉｍｅｒ（ＦＰ）
Ｏｕｔｅｒ　ｂｏｏｓｔ　ｐｒｉｍｅｒ（ｏＢＰ）
Ｔｕｒｎ－ｂａｃｋ　ｐｒｉｍｅｒ（ＴＰ），ｔｕｒｎ－ｂａｃｋ　ｓｉｔｅ
Ｉｎｔｅｒｎａｌ　ｂｏｏｓｔ　ｐｒｉｍｅｒ（ｉＢＰ）
Ｔｕｒｎ－ｂａｃｋ　ｐｒｉｍｅｒ（ＴＰ），ａｎｎｅａｌｉｎｇ　ｓｉｔｅ
Ｏｕｔｅｒ　ｐｒｉｍｅｒ（ＯＰ） Folding primer (FP)
Outer boost primer (oBP)
Turn-back primer (TP), turn-back site
Internal boost primer (iBP)
Turn-back primer (TP), annealing site
Outer primer (OP)
サンプルＲＮＡ
インターナルポジティブコントロールとして２＊１０５　Ｒｕｂｉｓｃｏ　ＲＮＡ　（ｎ＝３）を用いた。
目的核酸（ＲＮＡ）としてＳＡＲＳ－Ｃｏｖ－２のＲＮＡを用いた。 Sample RNA
As an internal positive control, 2*10 5 Rubisco RNA (n=3) was used.
SARS-Cov-2 RNA was used as the target nucleic acid (RNA).
増幅反応用バッファー
Ｔｒｉｚｍａ，　ｐＨ　８．０　２０　ｍＭ　、ＣＨ３ＣＯＯＫ　６０　ｍＭ、（ＮＨ４）２ＳＯ４　２０　ｍＭ、ＭｇＳＯ４　１６　ｍＭ、Ｔｗｅｅｎ　２０　０．２％　（ｗ／ｖ）、ｄＮＴＰ　１．４　ｍＭ、　ＲＮａｓｅ　ｆｒｅｅ－ｗａｔｅｒ） Amplification reaction buffer: Trizma, pH 8.0 20 mM, CH3COOK 60 mM, ( NH4 ) 2SO4 20 mM, MgSO4 16 mM, Tween 20 0.2% (w/v), dNTP 1.4 mM, RNase free water)
核酸増幅反応１
　反応槽１を用い、目的核酸（ＲＮＡ）の代わりに同量の水（ネガティブコントロール）と内部標準核酸（インターナルポジティブコントロール）のみを用いたＳｍａｒｔＡｍｐ法による核酸増幅（５回）を実施した。結果を図２に示す。内部標準核酸の増幅のみが確認された。 Nucleic acid amplification reaction 1
Nucleic acid amplification (five times) was performed by the SmartAmp method using reaction vessel 1, using the same amount of water (negative control) and only the internal standard nucleic acid (internal positive control) instead of the target nucleic acid (RNA). The results are shown in Figure 2. Only the amplification of the internal standard nucleic acid was confirmed.
核酸増幅反応２
　反応槽１を用い、目的核酸（ＲＮＡ）及び内部標準核酸のみを用いたＳｍａｒｔＡｍｐ法による核酸増幅（３回）を実施した。結果を図３に示す。内部標準核酸の増幅及び目的核酸の増幅（ポジティブコントロールと表示した３つのライン）が確認された。但し、図３には、目的核酸は用いず、内部標準核酸のみを用いたＳｍａｒｔＡｍｐ法による核酸増幅結果も示されている。 Nucleic acid amplification reaction 2
Nucleic acid amplification (three times) was performed by the SmartAmp method using only the target nucleic acid (RNA) and the internal standard nucleic acid in the reaction vessel 1. The results are shown in Figure 3. Amplification of the internal standard nucleic acid and the target nucleic acid (three lines indicated as positive control) were confirmed. However, Figure 3 also shows the results of nucleic acid amplification by the SmartAmp method using only the internal standard nucleic acid without using the target nucleic acid.
核酸増幅反応３
　反応槽２を用い、目的核酸（ＲＮＡ）の代わりに同量の水と内部標準核酸のみを用いたＳｍａｒｔＡｍｐ法による核酸増幅（６回）を実施した。結果を図４に示す。６回の内、３回の増幅反応で、内部標準核酸の増幅のみならず、その他の核酸増幅も確認され、偽陽性発生を示唆した。 Nucleic acid amplification reaction 3
Nucleic acid amplification (six times) was carried out by the SmartAmp method using reaction vessel 2, using the same amount of water and only the internal standard nucleic acid instead of the target nucleic acid (RNA). The results are shown in Figure 4. In three of the six amplification reactions, not only the amplification of the internal standard nucleic acid but also the amplification of other nucleic acids was confirmed, suggesting the occurrence of false positives.
核酸増幅反応４
　反応槽３を用い、目的核酸（ＲＮＡ）の代わりに同量の水と内部標準核酸のみを用いたＳｍａｒｔＡｍｐ法による核酸増幅（４回）を実施した。結果を図５に示す。内部標準核酸の増幅のみが確認された。 Nucleic acid amplification reaction 4
Nucleic acid amplification (four times) was carried out by the SmartAmp method using reaction vessel 3, using the same amount of water and only the internal standard nucleic acid instead of the target nucleic acid (RNA). The results are shown in Figure 5. Only the amplification of the internal standard nucleic acid was confirmed.
　以上の結果から、反応槽の内表面の表面粗さＲａが２５ｎｍ以下、好ましくは５～２０ｎｍの範囲、より好ましくは７～１７ｎｍの範囲、さらに好ましくは８～１５ｎｍの範囲であることが、偽陽性発生の原因と考えられる、目的核酸以外の増幅を抑制できることが分かる。 These results show that the surface roughness Ra of the inner surface of the reaction vessel is 25 nm or less, preferably in the range of 5 to 20 nm, more preferably in the range of 7 to 17 nm, and even more preferably in the range of 8 to 15 nm, which can suppress the amplification of nucleic acids other than the target nucleic acid, which is thought to be the cause of false positives.
　本発明は、核酸増幅及び検出に関連する分野において有用である。 The present invention is useful in fields related to nucleic acid amplification and detection.
１０　カートリッジ
２０　反応槽
２１　反応液収容用空間
２２　反応液供給用空間
２４　排気口
２５　突起部
３０，３１　流路
３２　反応液供給口
４０　フィルム 10 Cartridge 20 Reaction chamber 21 Reaction liquid storage space 22 Reaction liquid supply space 24 Exhaust port 25 Projection 30, 31 Flow path 32 Reaction liquid supply port 40 Film

Claims (13)

  1. 　核酸増幅用反応槽であって、反応槽の内表面の表面粗さＲａが２５ｎｍ以下である、前記反応槽。 A reaction vessel for nucleic acid amplification, the inner surface of which has a surface roughness Ra of 25 nm or less.
  2. 　前記反応槽は、反応槽本体と反応槽本体の開口を遮蔽するフィルムとで構成され、反応槽本体の内表面及びフィルムの表面粗さＲａが２５ｎｍ以下である、請求項１に記載の反応槽。 The reaction vessel according to claim 1, wherein the reaction vessel is composed of a reaction vessel body and a film that shields the opening of the reaction vessel body, and the surface roughness Ra of the inner surface of the reaction vessel body and the film is 25 nm or less.
  3. 　反応槽本体は、反応槽本体の内表面形成用表面の表面粗さＲａが２５ｎｍ以下である射出成形用型を用いて成形された射出成形品である、請求項１または２に記載の反応槽。 The reaction vessel according to claim 1 or 2, wherein the reaction vessel body is an injection molded product molded using an injection molding mold in which the surface roughness Ra of the inner surface forming surface of the reaction vessel body is 25 nm or less.
  4. 　前記反応槽は、反応槽の内表面にワックス被覆を有する射出成形品であり、ワックス被覆した内表面の表面粗さＲａが２５ｎｍ以下である、請求項１または２に記載の反応槽。 The reaction tank according to claim 1 or 2, wherein the reaction tank is an injection molded product having a wax coating on the inner surface of the reaction tank, and the surface roughness Ra of the wax-coated inner surface is 25 nm or less.
  5. 　前記反応槽は、反応液収容用空間を有し、
    　前記反応液収容用空間は、１０～５００μＬの範囲の容積を有する、請求項１～４のいずれか１項に記載の反応槽。     The reaction vessel has a space for containing a reaction liquid,
    The reaction vessel according to any one of claims 1 to 4, wherein the reaction liquid-accommodating space has a volume in the range of 10 to 500 µL.
  6. 　核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応である、請求項１～５のいずれか１項に記載の反応槽。 The reaction vessel according to any one of claims 1 to 5, wherein the nucleic acid amplification reaction is an isothermal amplification reaction or a thermocycle amplification reaction of nucleic acid.
  7. 　核酸の等温増幅反応は、核酸のＬＡＭＰ法またはＳｍａｒｔＡｍｐ法である、請求項６に記載の反応槽。 The reaction vessel according to claim 6, wherein the isothermal amplification reaction of nucleic acid is the LAMP method or the SmartAmp method of nucleic acid.
  8. 　核酸の等温増幅反応を、エキシトンプライマーまたはエキシトンプローブで標識検出する、請求項７に記載の反応槽。 The reaction vessel according to claim 7, in which an isothermal amplification reaction of a nucleic acid is detected by labeling with an exciton primer or an exciton probe.
  9. 　マイクロ流路で連絡されたチャンバーを有し、マイクロ流路（３０及び３１）を介して前記チャンバーと連絡する１つまたは２つ以上の核酸増幅用反応槽（２０）を含む核酸増幅用カートリッジ（１０）であって、
    　前記反応槽（２０）が、請求項１～８のいずれか１項に記載の反応槽である、前記カートリッジ。     A nucleic acid amplification cartridge (10) having chambers connected by a microchannel, the cartridge including one or more nucleic acid amplification reaction chambers (20) connected to the chambers via microchannels (30 and 31),
    The cartridge, wherein the reaction vessel (20) is a reaction vessel according to any one of claims 1 to 8.
  10. 　請求項９に記載のカートリッジを用い、前記カートリッジの反応槽中で、複数のプライマーを用いて目的核酸を増幅し、増幅した核酸を検出する、核酸の検出方法。 A method for detecting nucleic acid, comprising using the cartridge according to claim 9, amplifying a target nucleic acid in a reaction chamber of the cartridge using a plurality of primers, and detecting the amplified nucleic acid.
  11. 　核酸増幅反応は、核酸の等温増幅反応またはサーモサイクル増幅反応である、請求項１０に記載の核酸の検出方法。 The method for detecting nucleic acid according to claim 10, wherein the nucleic acid amplification reaction is an isothermal amplification reaction or a thermocycle amplification reaction of nucleic acid.
  12. 　核酸の等温増幅反応は、核酸のＬＡＭＰ法またはＳｍａｒｔＡｍｐ法である、請求項１１に記載の核酸の検出方法。 The method for detecting nucleic acid according to claim 11, wherein the isothermal amplification reaction of nucleic acid is the LAMP method or the SmartAmp method of nucleic acid.
  13. 　核酸の等温増幅反応を、エキシトンプライマーまたはエキシトンプローブで標識検出する、請求項１２に記載の核酸の検出方法。 The method for detecting nucleic acid according to claim 12, in which the isothermal amplification reaction of nucleic acid is detected by labeling with an exciton primer or an exciton probe.
PCT/JP2022/036723 2022-09-30 2022-09-30 Reaction vessel for nucleic acid amplification, cartridge, and nucleic acid detection method WO2024069938A1 (en)

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CN101096120A (en) * 2006-06-30 2008-01-02 深圳市群达行精密模具有限公司 Multi-part formed automobile inlet manifold injection mold and method
JP2010533490A (en) * 2007-07-13 2010-10-28 ハンディーラブ インコーポレイテッド Integrated device for nucleic acid extraction and diagnostic testing on multiple biological samples
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