WO2001030993A1 - Method of detecting target nucleic acid - Google Patents

Method of detecting target nucleic acid Download PDF

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Publication number
WO2001030993A1
WO2001030993A1 PCT/JP2000/007430 JP0007430W WO0130993A1 WO 2001030993 A1 WO2001030993 A1 WO 2001030993A1 JP 0007430 W JP0007430 W JP 0007430W WO 0130993 A1 WO0130993 A1 WO 0130993A1
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Prior art keywords
nucleic acid
primer
labeled
target nucleic
extension product
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PCT/JP2000/007430
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French (fr)
Japanese (ja)
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Yasumasa Mitani
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Wakunaga Pharmaceutical Co., Ltd.
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Priority to AU79558/00A priority Critical patent/AU7955800A/en
Publication of WO2001030993A1 publication Critical patent/WO2001030993A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method and a kit for specifically detecting a target nucleic acid in a biological sample.
  • PCR polymerase chain reaction method
  • RT-PCR RT-PCR
  • the gene amplification method represented by the reverse transcriptase polymerase chain reaction method has been widely studied and used as a method with high detection sensitivity.
  • a hybridization method in which a single or double strand having a base sequence complementary to a target nucleic acid is labeled with a radioactive or non-radioactive labeling substance and the target sequence is detected using complementarity with the target sequence is also known. It is widely used as a method for detecting the presence of a target nucleic acid or a target nucleic acid amplification product.
  • a liquid-solid hybridization method TR Gingeras et al., Nucleic Acid Res.
  • amplification of the target DNA or RNA can be performed in a short time, but detection of the amplified DNA fragment is complicated. However, it takes a long time until the hybridization method is performed. In the hybridization method, the operation is complicated and time-consuming, and a satisfactory method in terms of sensitivity and the like has not been obtained.
  • a method of amplifying a nucleic acid using a technique such as PCR using a first labeled nucleic acid primer as a sense primer and a second labeled nucleic acid primer labeled with a label different from the first labeled nucleic acid primer as an antisense primer has a disadvantage that when a non-specific amplification product other than the target nucleic acid amplification product is obtained, it is finally detected as a false positive. Disclosure of the invention
  • An object of the present invention is to provide a method and a kit for detecting a target nucleic acid quickly and with high sensitivity by a simple operation method.
  • the present inventors have conducted intensive studies on a method for detecting a nucleic acid that does not require a complicated hybridization reaction, and as a result, have found that a primer extension product labeled with two types of labels can be obtained. After that, it was found that the target nucleic acid could be detected simply, quickly and with high sensitivity by using affinity chromatography, and the present invention was completed. That is, the present invention relates to a method for specifically detecting a target nucleic acid in a biological material,
  • the present invention also provides a kit for specifically detecting a target nucleic acid in a biological material
  • the method for detecting a target nucleic acid according to the present invention comprises the above steps (a) to (d).
  • a known nucleic acid amplification reaction can be involved.
  • Such a nucleic acid amplification method is not particularly limited, and examples thereof include a ligase chain method (LCR) and a PCR method (Japanese Patent Application Laid-Open No. 62-281), with the PCR method being particularly preferred.
  • the primer set refers to a combination of a sense primer and a primer (antisense primer) complementary to a target nucleic acid sequence on the opposite side, and in the present invention, one or both of them are the first. It only needs to be labeled.
  • the first labeled nucleic acid primer and the second labeled nucleic acid primer used in the method for detecting a target nucleic acid of the present invention need to have different base sequences and have different labels.
  • the second labeled nucleic acid primer recognizes a part of a sequence other than the primer sequence in the extension product extended by the first labeled nucleic acid primer. As a result, only when annealing occurs, a double-stranded primer extension product having both labeled substances (both labeled primer extension products) is formed, and the target nucleic acid is detected.
  • the melting temperatures of the respective extension products will be different. By performing the annealing and extension reactions at the respective optimum annealing temperatures, both ends of the primer dimer and the like will be obtained. The generation of non-specific reaction by-products with different labels can be excluded.
  • the label must be one that specifically binds to the specific binding reagent present on the chromatography support.
  • Specific binding reagents and combinations of substances that specifically bind to it Examples of the combination include a combination of biotin and streptavidin or avidin, a hapten and an antibody, a ligand and a receptor, and the like. Among them, it is generally preferable to use an oligonucleotide having high heat stability and a small molecular size as the oligonucleotide.
  • the primer in the case of biotin and streptavidin, it is preferable to label the primer with biotin and apply the streptavidin to the chromatographic carrier, and the primer is attached to the chromatographic carrier by binding of the biotin and streptavidin. Join.
  • the label of either the first labeled nucleic acid primer or the second labeled nucleic acid primer must be capable of binding to a specific binding reagent immobilized at a predetermined position (detection region) of the chromatography carrier.
  • examples of such a label include DNP against an immobilized anti-DNP antibody.
  • nucleic acid used for the labeled nucleic acid primer a natural nucleic acid may be used as it is, or a synthetic oligonucleotide sequence may be used.
  • the position of the label in the primer may be any site that does not significantly affect the efficiency of the extension reaction of the primer, and preferably includes an active group in a hydroxyl group, a base, or a phosphodiester moiety near the 5 'end.
  • first labeled nucleic acid primer and the second labeled nucleic acid primer one labeled with a specific binding substance for detection such as biotin as the first labeled nucleic acid primer, and immobilized with DNP or the like as the second labeled nucleic acid primer
  • a primer labeled with a substance capable of binding to a reagent and having a sequence length different from that of the first labeled nucleic acid primer by 3 to 15 mer is selected.
  • Annealing by contacting the target nucleic acid with the first labeled nucleic acid primer and The extension reaction (step (a)) can be carried out under the conditions used for ordinary annealing.
  • the extension reaction is carried out at 30 to 80 ° C for 5 seconds, preferably at 35 to 72 for 10 to 60 seconds. You.
  • the primer extension product annealed and extended by the first labeling nucleic acid primer, and the nucleic acid amplification product amplified by the amplification reaction are complemented with ⁇ type.
  • This is performed using a typical second-labeled nucleic acid primer.
  • the target nucleic acid and the first labeled nucleic acid primer extension product in step (a) are used. It must be performed at a temperature lower than the melting temperature of
  • a double-stranded primer extension product having both labeled substances can be obtained, and the double-stranded nucleic acid can be used for the labeling of the first and second labeled nucleic acid primers.
  • a chromatographic carrier having a specific binding reagent such as an antibody that specifically binds
  • the presence / absence of the target nucleic acid can be detected simply, quickly and specifically. That is, in the method for detecting a target nucleic acid of the present invention, the target nucleic acid detected on the chromatography carrier is only a double-stranded nucleic acid having both labeled substances. If a specific amplification product is obtained, it will not be detected.
  • the so-called affinity mouth method is used for detection of both labeled primer extension products.
  • a specific binding reagent such as an antibody that binds to one of the double-stranded nucleic acids having both labels at a predetermined position (detection region) of the chromatography carrier is immobilized, and the other is used.
  • a specific binding reagent that can bind to a nucleic acid labeled product is kept in a dry state so that it can move when wet on the upstream side of the chromatography medium, and the target nucleic acid amplification product that has a labeled product at both ends is different. Is developed from the upstream of the chromatographic carrier by utilizing the capillary phenomenon.
  • the target nucleic acid extension and the specific binding reagent develop first while reacting, and finally are detected.
  • This is a method of capturing and detecting a target nucleic acid in the form of a sandwich-like complex in a region.
  • the target nucleic acid can be detected in an extremely short time as compared with the method of performing hybridization, for example, as short as 10 minutes or less as described in Examples below. .
  • the detection method is simple, and it does not require special techniques or equipment, so that multiple samples can be tested simultaneously. Moreover, the confirmation can be done visually and is extremely simple.
  • the chromatographic carrier used must have a pore size large enough to allow the target amplified nucleic acid to move stably and satisfactorily on the chromatographic carrier, to be sufficiently developed, and to reach the detection region without fail.
  • examples thereof include glass fiber filter paper, cellulose membrane, nitrocellulose membrane, and nylon membrane.
  • the specific binding reagent immobilized on the chromatography carrier is immobilized on a predetermined detection region, and binds to the target nucleic acid label in this region.
  • the specific binding reagent immobilized on the detection region include an antibody against hapten, for example, an anti-DNP antibody against DNP.
  • such an antibody may be either a monoclonal antibody or a polyclonal antibody.
  • the particles of the particulate specific binding reagent include colloid particles, inorganic particles, ceramic particles, metal or oxide particles thereof, gel particles, synthetic polymer particles, dye sol, and the like. From the viewpoint of dispersion stability, metal colloid particles such as gold colloid and synthetic polymer particles such as latex particles are particularly preferable.
  • the specific binding reagent is as described above, and avidin, streptavidin and the like are preferable.
  • the biological material used in the present invention may be any material as long as it contains the nucleic acid of interest. Examples of sources of such material include plants, insects, and animals. , Serum, plasma, cerebrospinal fluid, biopsy, tissue culture cells Examples thereof include a growth medium from tissue culture cells, a tissue extract, and a membrane washing solution.
  • the toxin type 1 gene (VT 1) of enterohemorrhagic Escherichia coli (E.co1i) was selected as the target nucleic acid.
  • a first labeled nucleic acid primer set and a second labeled nucleic acid primer as described below were used.
  • the primer on the sense side of the first labeled nucleic acid primer was a label capable of binding to colloidal gold-labeled streptavidin, and was introduced with a biotin at the 5 ′ end, while the primer on the antisense side was unlabeled.
  • DNP was introduced at the 5 ′ end as a label capable of binding to the immobilized anti-DNP antibody on the affinity-mouthed chromatography medium.
  • Imnodine ABC (Paull) was selected as a chromatographic carrier, and anti-DNP mouse monoclonal antibody was adjusted to 5 mg mL with 10 mM phosphate buffered saline, and dispenser Shotmaster II (Musashi Engineering) Then, it was coated in a line so as to have an I LZI cm, air-dried, gelatin-blocked, washed, and dried under negative pressure. The reaction film and the above-mentioned streptavidin pad labeled with colloidal gold were adhered on a tape to obtain an immunochromatography carrier. The obtained immunochromatographic medium was stored in an aluminum bag together with silica gel.
  • Enterohemorrhagic Escherichia coli with overnight cultured Vero toxin type 1 gene (VT 1) E. co 1 i
  • 1 X 1 0 6 with sterile water 1 X 1 0 5, 1 X 1 0 4, 1 X 1 It was adjusted to 0 3 ce 11 s ZmL.
  • These 100 were collected in 1.5 mL tubes, heated at 95 for 10 minutes, and subjected to nucleic acid amplification using 10 L of the solution as a target nucleic acid sample.
  • the target nucleic acid amplification product was detected by the following method. As a comparison, detection was performed by electrophoresis.
  • the above-mentioned second labeled nucleic acid primer 50 ng ZD, IL was added to the PCR reaction tube. Using a PCR device, heat denaturation was performed at 98 for 3 minutes, annealing at 65 for 30 seconds, and extension reaction at 72 for 30 seconds. Each reaction solution was developed on the immunochromatography carrier described above, and after 5 minutes, the presence or absence of color development was visually determined.
  • the method for detecting a target nucleic acid according to the present invention can greatly reduce the analysis time by performing an extension reaction using primers without performing a complicated operation in a so-called hybridization reaction.
  • the use of affinity chromatography makes it possible to analyze a sample simply, quickly and with high sensitivity.
  • the sample is in a roughly purified state and the sample can be easily prepared.
  • the primers can be completely adjusted to the target nucleic acid by appropriately adjusting the reaction conditions such as polymerase.
  • a distinction can be made between complementary and non-complementary cases. That is, point mutations can be easily detected without using the hybridization method or the like.

Abstract

A method of specifically detecting a target nucleic acid in a biological material characterized by comprising: (a) the step of bringing the target nucleic acid or a chain complementary thereto into contact with a first labeled nucleic acid primer complementary to either, or a primer set containing this primer to thereby form a primer extension product; (b) the step of denaturing the first nucleic acid primer extension product as described above to give a denatured chain; (c) the step of bringing this denatured chain into contact with a second labeled nucleic acid primer complementary thereto to form a both-labeled primer extension product; and (d) the step of developing this both-labeled primer extension product on a chromatographic carrier having specifically binding reagents which bind specifically to the labels of the first labeled nucleic acid primer and the second labeled nucleic acid primer; and a kit for detecting a target nucleic acid with the use of this method. Use of the detection method/detection kit makes it possible to conveniently, quickly and highly sensitively detect the presence or absence of the target nucleic acid in a sample.

Description

明 細 書 目的核酸の検出法 技術分野  Description Target nucleic acid detection method Technical field
本発明は、 生物試料中の目的核酸を特異的に検出するための方法及びキッ卜に 関する。 背景技術  The present invention relates to a method and a kit for specifically detecting a target nucleic acid in a biological sample. Background art
近年、 核酸の検出に関する研究や診断技術が急速に進歩し、 検査試料中の特定 遺伝子を検出する方法として、 遺伝子クローニング法、 サザンブロッテイング 法、 遺伝子増幅法及び八イブリダィゼーション法等いくつかの方法が知られてい る。  In recent years, research and diagnostic techniques related to the detection of nucleic acids have rapidly advanced, and several methods for detecting specific genes in test samples, such as gene cloning, Southern blotting, gene amplification, and octalization, have been developed. The method is known.
中でも、 PCR法 （polymerase chain reaction method) や RT— PCR法 Among them, PCR (polymerase chain reaction method) and RT-PCR
(reverse transcriptase polymerase chain reaction method) に代表される 遺伝子増幅法は、 検出感度が高い方法として広く研究及び利用されている。 また、 標的核酸と相補的な塩基配列をもつ単鎖又は二本鎖を放射性あるいは非 放射性の標識物質で標識し、 標的配列との相補性を利用して標的配列を検出する ハイブリダィゼ一ション法も、 目的核酸又は目的核酸増幅物の存在を検出するた めの方法として広く用いられている。 最近ではプローブを担体に固定する液相— 固相ハイブリダィゼーシヨン法 （T. R. Gingerasら： Nucleic Acid Res.15、 5373-5390等） やサンドイッチ型の液相一液相ハイブリダィゼーシヨン法 （Ann- Christine Syvaenenら： Nucleic Acid Res.14、 5037-5048(1986), 特開昭 62- 229068号公報等） も考案されている。 The gene amplification method represented by the reverse transcriptase polymerase chain reaction method has been widely studied and used as a method with high detection sensitivity. In addition, a hybridization method in which a single or double strand having a base sequence complementary to a target nucleic acid is labeled with a radioactive or non-radioactive labeling substance and the target sequence is detected using complementarity with the target sequence is also known. It is widely used as a method for detecting the presence of a target nucleic acid or a target nucleic acid amplification product. Recently, a liquid-solid hybridization method (TR Gingeras et al., Nucleic Acid Res. 15, 5373-5390, etc.) in which a probe is immobilized on a carrier, and a sandwich-type liquid-liquid one-liquid hybridization method (Ann-Christine Syvaenen et al .: Nucleic Acid Res. 14, 5037-5048 (1986), JP-A-62-229068, etc.) have also been devised.
しかし、 PCR法や RT— PCR法においては、 目的 DNA又は RNAの増幅 は短時間で行なえるものの、 増幅された DNA断片の検出操作が煩雑で検出する までに時間が掛かる等の問題があり、 ハイブリダィゼーション法においては操作 が煩雑で手間がかかり、 また感度等の点でも十分満足のいくものは得られていな い。 However, in the PCR and RT-PCR methods, amplification of the target DNA or RNA can be performed in a short time, but detection of the amplified DNA fragment is complicated. However, it takes a long time until the hybridization method is performed. In the hybridization method, the operation is complicated and time-consuming, and a satisfactory method in terms of sensitivity and the like has not been obtained.
また最近では、 検出感度の向上と操作の簡便性を目的として、 P C R法などの 遺伝子増幅法とハイプリダイゼーシヨン法を組み合わせた方法、 例えば P C尺で 増幅した標識 D N Aと固相に結合されたプローブ D N Aとで液相一固相間のハイ ブリダィゼーシヨン反応を行うことによって目的の核酸を検出する方法が報告さ れている （欧州特許公開 1 9 2 1 6 8号公報、 同 7 8 1 3 9号公報） 。 しかし、 当該検出法は、 多くは核酸の増幅と検出が別々の系で行われており、 その結果、 増幅により得られた産物が検出前に周囲の物質と接触することとなり、 交差汚染 や偽陽性結果を招く危険性があった。 また、 目的核酸の検出には核酸の分離を必 要とし、 分析にかなりの時間を要していた。  Recently, for the purpose of improving detection sensitivity and simplicity of operation, a method combining a gene amplification method such as PCR and a hybridization method, for example, a method in which labeled DNA amplified by a PC scale is bound to a solid phase and A method for detecting a target nucleic acid by carrying out a hybridization reaction between a liquid phase and a solid phase with a probe DNA has been reported (European Patent Publication No. 192 168, 7th edition). No. 8139). However, most of the detection methods use separate systems for amplification and detection of nucleic acids, and as a result, the products obtained by amplification come into contact with surrounding substances before detection, resulting in cross-contamination or false contamination. There was a risk of giving a positive result. Also, the detection of the target nucleic acid required the separation of the nucleic acid, and the analysis required a considerable amount of time.
更に、 第一標識核酸プライマーをセンスプライマーとし、 第一標識核酸プライ マーと異なる標識物で標識された第二標識核酸プライマーをアンチセンスプライ マーとして P C R等の手法を用いて核酸の増幅を行う方法も報告されているが、 当該方法では目的とする核酸増幅物以外に非特異的な増幅物が得られた場合に、 それが最終的に擬陽性として検出されてしまうという欠点があつた。 発明の開示  Furthermore, a method of amplifying a nucleic acid using a technique such as PCR using a first labeled nucleic acid primer as a sense primer and a second labeled nucleic acid primer labeled with a label different from the first labeled nucleic acid primer as an antisense primer. However, this method has a disadvantage that when a non-specific amplification product other than the target nucleic acid amplification product is obtained, it is finally detected as a false positive. Disclosure of the invention
本発明の目的は、 簡便な操作方法で、 迅速且つ高感度で目的核酸を検出するた めの方法及びキットを提供することにある。  An object of the present invention is to provide a method and a kit for detecting a target nucleic acid quickly and with high sensitivity by a simple operation method.
本発明者らは、 斯かる実状に鑑み、 操作が複雑なハイブリダィゼーシヨン反応 を必要としない核酸の検出法について鋭意検討した結果、 二種類の標識物で標識 されたプライマー伸長生成物を得た後、 ァフィ二ティークロマトグラフィーを利 用することにより、 簡便 ·迅速且つ高感度に目的核酸を検出できることを見出 し、 本発明を完成するに至った。 即ち、 本発明は、 生物材料中の目的核酸を特異的に検出する方法であって、In view of such circumstances, the present inventors have conducted intensive studies on a method for detecting a nucleic acid that does not require a complicated hybridization reaction, and as a result, have found that a primer extension product labeled with two types of labels can be obtained. After that, it was found that the target nucleic acid could be detected simply, quickly and with high sensitivity by using affinity chromatography, and the present invention was completed. That is, the present invention relates to a method for specifically detecting a target nucleic acid in a biological material,
( a ) 目的核酸又はその相補鎖に、 それらのいずれかと相補的な第一標識核酸 プライマー又は該プライマーを含むプライマーセットを接触させてプライマー伸 長生成物を形成させる工程、 (a) contacting a target nucleic acid or its complementary strand with a first labeled nucleic acid primer complementary to any of them or a primer set containing the primer to form a primer extension product,
( b ) 該第一核酸プライマ一伸長生成物を変性させて変性鎖とする工程、 (b) denaturing the first nucleic acid primer extension product into a denatured strand,
( c ) 該変性鎖に、 これと相補的な第二標識核酸プライマーを接触させて両標 識プライマー伸長生成物を形成させる工程、 (c) contacting the modified strand with a second labeled nucleic acid primer complementary thereto to form an extension product of both labeled primers,
( d ) 両標識プライマー伸長生成物を、 第一標識核酸プライマー及び第二標識 核酸プライマーの標識と特異的に結合する特異結合試薬を有するクロマ卜グラフ ィ一担体に展開させる工程、  (d) developing both labeled primer extension products on a chromatographic carrier having a specific binding reagent that specifically binds to the labels of the first labeled nucleic acid primer and the second labeled nucleic acid primer;
を行うことを特徴とする目的核酸の検出法を提供するものである。 And a method for detecting a target nucleic acid.
また本発明は、 生物材料中の目的核酸を特異的に検出するキッ卜であって、 The present invention also provides a kit for specifically detecting a target nucleic acid in a biological material,
( a ) 目的核酸又はその相補鎖に、 それらのいずれかと相補的な第一標識核酸 プライマー又は該プライマーを含むプライマーセットを接触させてプライマー伸 長生成物を形成させる手段、 (a) a means for forming a primer extension product by contacting a target nucleic acid or a complementary strand thereof with a first labeled nucleic acid primer complementary to any of them or a primer set containing the primer,
( b ) 該第一核酸プライマー伸長生成物を変性させて変性鎖とする手段、 (b) means for denaturing the first nucleic acid primer extension product into a denatured strand,
( c ) 該変性鎖に、 これと相補的な第二標識核酸プライマ一を接触させて両標 識プライマー伸長生成物を形成させる手段、 (c) means for contacting the denatured strand with a second labeled nucleic acid primer complementary thereto to form an extension product of both labeled primers,
( d ) 該両標識プライマー伸長生成物を、 第一標識核酸プライマー及び第二標 識核酸プライマーの標識と特異的に結合する特異結合試薬を有するクロマトダラ フィ一担体に展開させる手段、  (d) means for developing the two labeled primer extension products on a chromatographic carrier having a specific binding reagent that specifically binds to the labels of the first labeled nucleic acid primer and the second labeled nucleic acid primer;
からなる目的核酸の検出キットを提供するものである。 発明を実施するための最良の形態 And a kit for detecting the target nucleic acid. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の目的核酸の検出法は、 上記工程 （a ) 〜 （d ) からなるものである が、 より高感度の検出が求められる場合、 例えば特に検出対象の塩基配列の量が 少ない時は、 公知の核酸増幅反応を伴うことができる。 斯かる核酸増幅法として は特に限定されるものではないが、 リガーゼ連鎖法 （L C R) や P C R法 （特開 昭 62- 281号公報） が挙げられ、 特に P C R法が好ましい。 例えば P C R法を用い る場合は、 P C R試薬の存在下、 （a ) 第一標識核酸プライマーを含むプライマ 一セットを接触させてプライマー伸長生成物を形成させる工程と、 （b ) 該第一 標識核酸プライマー伸長生成物を変性させて変性鎖とする工程を、 例えば 2 0〜 4 0サイクル繰り返し行なうことにより標識核酸を増幅することができる。 ここで、 プライマーセットとは、 センスプライマーと、 反対側の目的核酸配列 に相補的なプライマー （アンチセンスプライマー） との組み合せをいい、 本発明 においてはこれらのうちのいずれか一方又は双方が第一標識されていればよい。 本発明の目的核酸の検出法において用いられる第一標識核酸プライマーと第二 標識核酸プライマ一は、 それぞれ異なる塩基配列を有し、 且つ異なる標識をもつ ことが必要である。 The method for detecting a target nucleic acid according to the present invention comprises the above steps (a) to (d). When the amount is small, a known nucleic acid amplification reaction can be involved. Such a nucleic acid amplification method is not particularly limited, and examples thereof include a ligase chain method (LCR) and a PCR method (Japanese Patent Application Laid-Open No. 62-281), with the PCR method being particularly preferred. For example, when using the PCR method, (a) a step of contacting a set of primers containing a first labeled nucleic acid primer to form a primer extension product in the presence of a PCR reagent; The labeled nucleic acid can be amplified by repeating the step of denaturing the primer extension product to form a denatured strand, for example, 20 to 40 cycles. Here, the primer set refers to a combination of a sense primer and a primer (antisense primer) complementary to a target nucleic acid sequence on the opposite side, and in the present invention, one or both of them are the first. It only needs to be labeled. The first labeled nucleic acid primer and the second labeled nucleic acid primer used in the method for detecting a target nucleic acid of the present invention need to have different base sequences and have different labels.
両プライマ一の塩基配列が異なるものとすることより、 第二標識核酸プライマ 一が、 第一標識核酸プライマ一により伸長された伸長生成物のうち該プライマ一 配列以外の配列の一部を認識することによってアニーリングが起った場合にのみ 両標識物を有する 2本鎖状態のプライマー伸長生成物 （両標識プライマー伸長生 成物） が形成され、 目的の核酸が検出されることになる。  By making the base sequences of both primers different, the second labeled nucleic acid primer recognizes a part of a sequence other than the primer sequence in the extension product extended by the first labeled nucleic acid primer. As a result, only when annealing occurs, a double-stranded primer extension product having both labeled substances (both labeled primer extension products) is formed, and the target nucleic acid is detected.
斯かる両プライマ一を用いれば、 それぞれの伸長生成物の融解温度が異なるも のとなり、 それぞれの至適ァニーリング温度でァニ一リング及び伸長反応を行う ことにより、 プライマーダイマ一等の両末端が異なる標識物をもつ非特異的な反 応副生成物が生成されることを排除できる。 尚、 第一標識核酸プライマーと第二 標識核酸プライマーの配列長に、 3〜2 0 m e r、 好ましくは 3〜1 5 m e rの 差を設けることが、 ァニーリングの選択性を高める上で好ましい。  If such primers are used, the melting temperatures of the respective extension products will be different. By performing the annealing and extension reactions at the respective optimum annealing temperatures, both ends of the primer dimer and the like will be obtained. The generation of non-specific reaction by-products with different labels can be excluded. In addition, it is preferable to provide a difference of 3 to 20 m er, preferably 3 to 15 m er in the sequence length between the first labeled nucleic acid primer and the second labeled nucleic acid primer in order to enhance the selectivity of annealing.
標識は、 クロマトグラフィー担体上に存在する特異結合試薬と特異的に結合す るものである必要がある。 特異結合試薬及びそれと特異結合する物質の組み合わ せとしては、 例えば、 ピオチンとストレプトアビジン或いはアビジン、 ハプテン と抗体、 リガンドとレセプ夕一などの組み合わせが挙げられる。 このうち、 一般 的にはオリゴヌクレオチドの方に、 熱に対して安定性の高く、 分子の大きさの小 さいものを用いることが好ましい。 例えば、 ピオチンとストレプトアビジンの場 合にはプライマ一にピオチン標識を行ない、 クロマトグラフィー担体側にストレ ブトアビジンを塗布させておくことが好ましく、 プライマ一はピオチンとストレ ブトアビジンの結合によってクロマトグラフィー担体上に結合する。 The label must be one that specifically binds to the specific binding reagent present on the chromatography support. Specific binding reagents and combinations of substances that specifically bind to it Examples of the combination include a combination of biotin and streptavidin or avidin, a hapten and an antibody, a ligand and a receptor, and the like. Among them, it is generally preferable to use an oligonucleotide having high heat stability and a small molecular size as the oligonucleotide. For example, in the case of biotin and streptavidin, it is preferable to label the primer with biotin and apply the streptavidin to the chromatographic carrier, and the primer is attached to the chromatographic carrier by binding of the biotin and streptavidin. Join.
また、 第一標識核酸プライマーと第二標識核酸プライマーの何れか一方の標識 は、 クロマトグラフィー担体の所定の位置 （検出領域） に固相化された特異結合 試薬と結合可能なものである必要があり、 斯かる標識としては例えば固相化抗 D N P抗体に対する D N Pが挙げられる。  Further, the label of either the first labeled nucleic acid primer or the second labeled nucleic acid primer must be capable of binding to a specific binding reagent immobilized at a predetermined position (detection region) of the chromatography carrier. Yes, examples of such a label include DNP against an immobilized anti-DNP antibody.
これらピオチン、 ハプテン、 リガンド等の標識物は、 いずれも単独、 或いは必 要があれば複数の組み合わせで公知の手段 （特開昭 5 9 - 9 3 0 9 9号、 同 5 9 一 1 4 8 7 9 8号、 同 5 9— 2 0 4 2 0 0号各公報参照） により導入することが できる。  These labeled substances such as biotin, hapten, ligand and the like may be used alone or, if necessary, in combination of a plurality of known methods (Japanese Patent Application Laid-Open Nos. 59-93099 and 591-148). No. 798, No. 59-204, 2004).
標識核酸プライマーに用いる核酸は、 天然の核酸をそのまま使用してもよく、 また合成オリゴヌクレオチド配列を利用しても良い。 プライマー中の標識物の位 置はプライマーの伸長反応の効率に大きく影響を与えないところであればよく、 好ましくは 5 '末端付近の水酸基部分、 塩基部分或いはリン酸ジエステル部分の 活性基が挙げられる。  As the nucleic acid used for the labeled nucleic acid primer, a natural nucleic acid may be used as it is, or a synthetic oligonucleotide sequence may be used. The position of the label in the primer may be any site that does not significantly affect the efficiency of the extension reaction of the primer, and preferably includes an active group in a hydroxyl group, a base, or a phosphodiester moiety near the 5 'end.
第一標識核酸プライマー及び第二標識核酸プライマーの好ましい態様として は、 第一標識核酸プライマーとしてピオチン等の検出用特異結合物質で標識され たものを、 第二標識核酸プライマーとして D N P等の固相化試薬と結合可能な物 質で標識され、 その配列長が第一標識核酸プライマーより 3〜1 5 m e r異なる ものを選択する場合が挙げられる。  As a preferred embodiment of the first labeled nucleic acid primer and the second labeled nucleic acid primer, one labeled with a specific binding substance for detection such as biotin as the first labeled nucleic acid primer, and immobilized with DNP or the like as the second labeled nucleic acid primer There is a case where a primer labeled with a substance capable of binding to a reagent and having a sequence length different from that of the first labeled nucleic acid primer by 3 to 15 mer is selected.
目的核酸と第一標識核酸プライマーを接触させることによるアニーリング及び 伸長反応 （工程 （a ) ) は、 通常のアニーリングに用いられる条件が適用でき、 例えば、 3 0〜 8 0 °Cで 5秒、 好ましくは 3 5〜 7 2 で 1 0〜 6 0秒行なわれ る。 Annealing by contacting the target nucleic acid with the first labeled nucleic acid primer and The extension reaction (step (a)) can be carried out under the conditions used for ordinary annealing. For example, the extension reaction is carried out at 30 to 80 ° C for 5 seconds, preferably at 35 to 72 for 10 to 60 seconds. You.
また、 第二標識核酸プライマーを用いるアニーリング及び伸長反応は、 第一標 識核酸プライマ一によってァニーリング及び伸長されたプライマー伸長生成物、 更には増幅反応によって増幅された核酸増幅物を铸型としてそれと相補的な第二 標識核酸プライマーにより行われる。 斯かる反応は、 第一標識核酸プライマーと して第二標識核酸プライマーより長い配列長を有するものを用いた場合には、 ェ 程 （a ) における目的核酸と第一標識核酸プライマー伸長生成物との融解温度よ り低レ、温度で行うことが必要である。  In the annealing and extension reaction using the second labeled nucleic acid primer, the primer extension product annealed and extended by the first labeling nucleic acid primer, and the nucleic acid amplification product amplified by the amplification reaction are complemented with 铸 type. This is performed using a typical second-labeled nucleic acid primer. In such a reaction, when a primer having a longer sequence length than the second labeled nucleic acid primer is used as the first labeled nucleic acid primer, the target nucleic acid and the first labeled nucleic acid primer extension product in step (a) are used. It must be performed at a temperature lower than the melting temperature of
かくして、 両標識物を有する 2本鎖状態のプライマー伸長生成物 （両標識ブラ イマ一伸長生成物） を得ることができ、 その 2本鎖核酸を、 第一、 第二標識核酸 プライマーの標識と特異に結合する抗体などの特異結合試薬を有するクロマトグ ラフィ一担体に展開させることにより、 簡便 ·迅速且つ特異的に目的核酸の有無 を検知することをことができる。 即ち、 本発明の目的核酸の検出法においては、 クロマトグラフィ一担体上で検出される目的核酸は、 両標識物を有する 2本鎖核 酸のみであることから、 目的とする核酸増幅物以外に非特異的な増幅物が得られ た場合においても、 それが検出されることはない。  Thus, a double-stranded primer extension product having both labeled substances (both labeled primer-primed extension product) can be obtained, and the double-stranded nucleic acid can be used for the labeling of the first and second labeled nucleic acid primers. By developing on a chromatographic carrier having a specific binding reagent such as an antibody that specifically binds, the presence / absence of the target nucleic acid can be detected simply, quickly and specifically. That is, in the method for detecting a target nucleic acid of the present invention, the target nucleic acid detected on the chromatography carrier is only a double-stranded nucleic acid having both labeled substances. If a specific amplification product is obtained, it will not be detected.
両標識プライマー伸長生成物の検出は、 いわゆるァフィ二ティーク口マト法が 用いられる。 具体的には、 クロマトグラフィー担体の所定の位置 （検出領域） に 両標識物を有する 2本鎖核酸のうちの一方の標識物と結合する抗体等の特異結合 試薬を固相化し、 更にもう一方の核酸標識物と結合することのできる特異結合試 薬をクロマトダラフ媒体のより上流側に湿潤時に移動可能なように乾燥状態で保 持しておき、 両末端が異なる標識物をもつ目的核酸増幅物を含む試料を上記クロ マトグラフィ一担体の上流より毛細管現象を利用して展開させるものである。 即 ち、 最初に目的核酸伸長物と特異結合試薬が反応しながら展開し、 最終的に検出 領域でサンドィツチ状複合体の形で、 目的核酸を捕獲して検出する方法である。 当該ァフィニティーク口マトグラフィ一担体の利用により、 ハイブリダイゼ一 シヨンを行う方法に比べて極めて短い時間、 例えば後記実施例で示すように 1 0 分以内の短い時間で目的核酸の検出を行なうことができる。 The so-called affinity mouth method is used for detection of both labeled primer extension products. Specifically, a specific binding reagent such as an antibody that binds to one of the double-stranded nucleic acids having both labels at a predetermined position (detection region) of the chromatography carrier is immobilized, and the other is used. A specific binding reagent that can bind to a nucleic acid labeled product is kept in a dry state so that it can move when wet on the upstream side of the chromatography medium, and the target nucleic acid amplification product that has a labeled product at both ends is different. Is developed from the upstream of the chromatographic carrier by utilizing the capillary phenomenon. That is, the target nucleic acid extension and the specific binding reagent develop first while reacting, and finally are detected. This is a method of capturing and detecting a target nucleic acid in the form of a sandwich-like complex in a region. By using the affinity mouth chromatography carrier, the target nucleic acid can be detected in an extremely short time as compared with the method of performing hybridization, for example, as short as 10 minutes or less as described in Examples below. .
また、 その検出方法も簡便であり、 特殊な技術や装置を必要とせず、 多検体を 同時に検査することもできる。 しかもその確認は、 視覚的に行なうこともでき極 めて簡単である。  In addition, the detection method is simple, and it does not require special techniques or equipment, so that multiple samples can be tested simultaneously. Moreover, the confirmation can be done visually and is extremely simple.
ここで、 用いられるクロマトグラフィー担体は、 目的増幅核酸が安定かつ良好 にクロマトグラフィー担体上を移動して十分な展開がなされ、 検出領域に確実に 到達できるような大きさのポアサイズを有することが必要であり、 例えば、 ガラ ス繊維濾紙、 セルロース膜、 ニトロセルロース膜、 ナイロン膜等が挙げられる。 クロマトグラフィー担体上に固相化される特異的結合試薬は、 あらかじめ決め られた検出領域に固相化され、 この領域で目的核酸標識物と結合する。 斯かる検 出領域に固相化されている特異結合試薬としては、 ハプテンに対する抗体、 例え ば D N Pに対する抗 D N P抗体等が挙げられる。 尚、 斯かる抗体はモノクロ一ナ ル抗体、 ポリクローナル抗体のいずれであっても構わない。  Here, the chromatographic carrier used must have a pore size large enough to allow the target amplified nucleic acid to move stably and satisfactorily on the chromatographic carrier, to be sufficiently developed, and to reach the detection region without fail. Examples thereof include glass fiber filter paper, cellulose membrane, nitrocellulose membrane, and nylon membrane. The specific binding reagent immobilized on the chromatography carrier is immobilized on a predetermined detection region, and binds to the target nucleic acid label in this region. Examples of the specific binding reagent immobilized on the detection region include an antibody against hapten, for example, an anti-DNP antibody against DNP. In addition, such an antibody may be either a monoclonal antibody or a polyclonal antibody.
一方、 クロマトグラフィー担体上に移動可能な状態で保持される特異結合試薬 としては、 粒子状の特異結合試薬であり且つ直接目視可能な物質が好ましい。 こ こで粒子状特異結合試薬の粒子としては、 コロイド粒子、 無機粒子、 セラミック 粒子、 金属、 またはその酸化物粒子、 ゲル状粒子、 合成高分子粒子、 染料ゾルな どが挙げられるが、 粒子の分散安定性の点で特に金コロイドなどの金属コロイド 粒子やラテックス粒子などの合成高分子粒子が好ましい。 尚、 特異結合試薬とし ては前記したとおりであり、 アビジン、 ストレプトアビジン等が好ましい。 尚、 本発明に用いる生物材料は、 目的核酸を含んでいればどのようなものであ つてもよく、 斯かる材料の採取源としては、 植物、 昆虫および動物が挙げられ、 好ましい生物材料としては、 血清、 血漿、 髄液、 生検試料、 組織培養細胞若しく は組織培養細胞からの増殖培地、 組織抽出物又は膜洗浄液等が挙げられる。 実施例 On the other hand, as the specific binding reagent retained in a state capable of moving on the chromatography carrier, a substance which is a particulate specific binding reagent and is directly visible is preferable. Here, the particles of the particulate specific binding reagent include colloid particles, inorganic particles, ceramic particles, metal or oxide particles thereof, gel particles, synthetic polymer particles, dye sol, and the like. From the viewpoint of dispersion stability, metal colloid particles such as gold colloid and synthetic polymer particles such as latex particles are particularly preferable. The specific binding reagent is as described above, and avidin, streptavidin and the like are preferable. The biological material used in the present invention may be any material as long as it contains the nucleic acid of interest. Examples of sources of such material include plants, insects, and animals. , Serum, plasma, cerebrospinal fluid, biopsy, tissue culture cells Examples thereof include a growth medium from tissue culture cells, a tissue extract, and a membrane washing solution. Example
以下に、 実施例を挙げて本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail with reference to examples.
参考例 1 標識核酸プライマーの調製  Reference Example 1 Preparation of labeled nucleic acid primer
目的核酸として腸管出血性大腸菌 （E. c o 1 i ) のべ口毒素 1型遺伝子 (VT 1) を選択した。 下記の様な第一標識核酸プライマ一セットと第二標識核 酸プライマ一を使用した。  The toxin type 1 gene (VT 1) of enterohemorrhagic Escherichia coli (E.co1i) was selected as the target nucleic acid. A first labeled nucleic acid primer set and a second labeled nucleic acid primer as described below were used.
(1) 第一標識核酸プライマーセット  (1) First labeled nucleic acid primer set
B i o t i n- 5' T TT A C C T T A G A C TT C T C G A C 3 ' (配列番号 1)  B i o t i n- 5 'T TT A C C T T A G A C TT C T C G A C 3' (SEQ ID NO: 1)
5' CACATATAAATTATTTCGTTC 3' (配列番号 2)  5 'CACATATAAATTATTTCGTTC 3' (SEQ ID NO: 2)
(2) 第二標識核酸プライマー  (2) Second labeled nucleic acid primer
3' (配列番号 3) 3 '(SEQ ID NO: 3)
第一標識核酸プライマーのセンス側のプライマーは、 金コロイド標識ストレブ トァビジンに結合可能な標識物として、 5' 末端にピオチンを導入し、 アンチ センス側のプライマーは非標識とした。  The primer on the sense side of the first labeled nucleic acid primer was a label capable of binding to colloidal gold-labeled streptavidin, and was introduced with a biotin at the 5 ′ end, while the primer on the antisense side was unlabeled.
第二標識核酸プライマーは、 ァフィ二ティーク口マトグラフ媒体上の固相化抗 DNP抗体に結合可能な標識物として、 5' 末端に DNPを導入した。  As the second labeled nucleic acid primer, DNP was introduced at the 5 ′ end as a label capable of binding to the immobilized anti-DNP antibody on the affinity-mouthed chromatography medium.
参考例 2 金コロイド標識ストレプトアビジンを吸収させたパッ卜の調製 金コロイド溶液 lmL (BBI社製；粒径 40 m) にストレプトアビジン 0. lmgを添加し、 5分間攪拌した。 10%BSA (pH9. 0) を 0. 1 mL加え、 10分間攪拌し、 ブロッキングを行なった後、 1%BSA、 0. 05 %PEG20, 000を含む 2 OmMトリス塩酸緩衝液 （pH8. 2) で 3回遠 心洗浄し、 沈査を 1%BSA、 0. 05 %PEG 20, 000を含む 20mM P T/JP00/07430 トリス塩酸緩衝液 （pH 8. 2) で再浮遊させ、 5%BSA、 0. 05 %PEG 2 0， 0 0 0、 0. 3 M N a C 1を含む 2 OmMトリス塩酸緩衝液 （pH 8. 2) で 2倍希釈した。 得られた金コロイド標識化ストレプトアビジンを波長 52011111で吸光度が1. 0になるように、 3%BSA、 0. 05 %PEG 2 0， 0 0 0、 0. 15M N a C 1を含む 2 OmMトリス塩酸緩衝液 （pH 8. 2) で希釈した。 金コロイド標識ストレプトアビジンをコンジユゲートパッ 卜に吸収させ、 ー晚凍結乾燥させ、 金コロイド標識ストレプトアビジンのパット 調製を行なった。 Reference Example 2 Preparation of a Pad Absorbing Streptavidin Labeled with Colloidal Gold Streptavidin (0.1 mg) was added to 1 mL of colloidal gold solution (BBI; particle size: 40 m) and stirred for 5 minutes. Add 0.1 mL of 10% BSA (pH 9.0), stir for 10 minutes, perform blocking, and then add 2% OmM Tris-HCl buffer (pH 8.2) containing 1% BSA and 0.05% PEG20,000. Wash 3 times with centrifugation, and settle down to 20 mM containing 1% BSA, 0.05% PEG 20,000 PT / JP00 / 07430 Resuspend in Tris-HCl buffer (pH 8.2) and contain 2% OmM Tris-HCl buffer containing 5% BSA, 0.05% PEG20, 00, 0,3 MNaCl The solution (pH 8.2) was diluted twice. The obtained colloidal gold-labeled streptavidin was treated with 2 OmM containing 3% BSA, 0.05% PEG20, 0.000, 0.15M NaC1 so that the absorbance at wavelength 52011111 was 1.0. It was diluted with Tris-HCl buffer (pH 8.2). The colloidal gold-labeled streptavidin was absorbed into a conjugation pad and lyophilized to prepare a pad of colloidal gold-labeled streptavidin.
参考例 3 ァフィ二ティークロマトグラフィー担体の調製  Reference Example 3 Preparation of affinity chromatography support
クロマトグラフィー担体としてィムノダイン ABC (P au l社） を選び、 抗 DNPマウスモノクローナル抗体を 10 mMリン酸緩衝液生理食塩水で、 5mg mLに調製し、 ディスペンサー装置ショットマスター II (武蔵エンジニアリン グ社） を用い、 I LZI cmとなるようにライン状に塗布し、 自然乾燥させた 後、 ゼラチンブロッキング後、 洗浄し、 陰圧乾燥させた。 テープ上に反応膜と前 記の金コロイド標識ストレプトアビジンパッドを貼り付け、 免疫クロマトグラフ ィー担体とした。 得られた免疫クロマトグラフ媒体はシリカゲルと共にアルミ袋 に保存した。  Imnodine ABC (Paull) was selected as a chromatographic carrier, and anti-DNP mouse monoclonal antibody was adjusted to 5 mg mL with 10 mM phosphate buffered saline, and dispenser Shotmaster II (Musashi Engineering) Then, it was coated in a line so as to have an I LZI cm, air-dried, gelatin-blocked, washed, and dried under negative pressure. The reaction film and the above-mentioned streptavidin pad labeled with colloidal gold were adhered on a tape to obtain an immunochromatography carrier. The obtained immunochromatographic medium was stored in an aluminum bag together with silica gel.
実施例 1 目的核酸の検出  Example 1 Detection of target nucleic acid
一夜培養したベロ毒素 1型遺伝子 （VT 1) を有する腸管出血性大腸菌 （E. c o 1 i ) を滅菌水で 1 X 1 06、 1 X 1 05、 1 X 1 04、 1 X 1 03c e 1 1 s ZmLに調製した。 それら 100 を 1. 5mLチューブに分取し、 95 で 10分間加熱した後、 その溶液 10 Lを目的核酸試料として核酸増幅に供し た。 Enterohemorrhagic Escherichia coli with overnight cultured Vero toxin type 1 gene (VT 1) (E. co 1 i) 1 X 1 0 6 with sterile water, 1 X 1 0 5, 1 X 1 0 4, 1 X 1 It was adjusted to 0 3 ce 11 s ZmL. These 100 were collected in 1.5 mL tubes, heated at 95 for 10 minutes, and subjected to nucleic acid amplification using 10 L of the solution as a target nucleic acid sample.
上記の第一標識核酸プライマ一をそれぞれ 5 Ong用いて、 40 /Lの 10 mMトリス—塩酸緩衝液 （pH8. 5) 、 5 OmM KCし 1. 5mM Mg C 12、 各 2 0 0 の d ATP， d GTP, dCTP, dTTP、 1. 25 u n i tの Taq D N Aポリメラ一ゼを含む溶液および前記の目的核酸試料 10 Lをそれぞれ同一の PC R用チューブに入れ、 PCR法による増幅反応を 行なった。 この反応は、 94°Cで 10分間加熱後、 94°C, 30秒、 50°C, 30 秒、 72 °C, 30秒のサイクルで 40回繰り返した。 It said first labeled nucleic primers scratch using each 5 Ong, 40/10 mM Tris L - HCl buffer (. PH8 5), 5 OmM KC and 1. 5mM Mg C 1 2, each 2 0 0 d ATP, d GTP, dCTP, dTTP, 1.25 A solution containing the unit Taq DNA polymerase and 10 L of the target nucleic acid sample were placed in the same PCR tube, and an amplification reaction was performed by PCR. This reaction was heated at 94 ° C for 10 minutes and then repeated 40 times in a cycle of 94 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, 30 seconds.
目的核酸増幅物を以下の方法で検出した。 尚、 比較として電気泳動法による検 出を行なった。  The target nucleic acid amplification product was detected by the following method. As a comparison, detection was performed by electrophoresis.
検出法 1 ：  Detection method 1:
前記の PCR反応チューブ中に、 上記の第二標識核酸プライマー （50ngZ D を I L添加した。 PCR装置を用い、 98 で 3分間熱変性、 65 で 30秒間ァニーリング、 72 で 30秒間伸長反応を行なった。 各反応液を上記 の免疫クロマトグラフィー担体に展開させ、 5分後に発色の有無を目視判定し た。 結果を表 1に示す。  The above-mentioned second labeled nucleic acid primer (50 ng ZD, IL was added to the PCR reaction tube. Using a PCR device, heat denaturation was performed at 98 for 3 minutes, annealing at 65 for 30 seconds, and extension reaction at 72 for 30 seconds. Each reaction solution was developed on the immunochromatography carrier described above, and after 5 minutes, the presence or absence of color development was visually determined.
検出法 2 (比較例） ：  Detection method 2 (Comparative example):
ァガロースゲルを用い、 100Vで 30分間電気泳動後、 ェチジゥムブ口マイ ド溶液で染色後、 紫外線照射により、 目的核酸増幅物のバンドを確認した。 結果 を表 1に併せて示す。  After electrophoresis at 100 V for 30 minutes using an agarose gel, the cells were stained with an ethidium solution, and the band of the target nucleic acid amplification product was confirmed by ultraviolet irradiation. The results are also shown in Table 1.
Figure imgf000012_0001
Figure imgf000012_0001
+ + シグナルの発色が強  + + Strong signal color
+ シグナルの発色が有  + Signal color
シグナルの発色がなし  No signal coloring
十 シグナルの発色が不明瞭 P /JP00/07430 表 1に示すように、 本発明の目的核酸の検出法は、 電気泳動法と同程度の感度 での検出が可能であった。 産業上の利用可能性 (10) Signal coloring is unclear P / JP00 / 07430 As shown in Table 1, the target nucleic acid detection method of the present invention was able to be detected with the same sensitivity as the electrophoresis method. Industrial applicability
本発明による目的核酸の検出法は、 いわゆるハイブリダィゼーション反応にお ける煩雑な操作を行なわないでプライマーによる伸長反応を行なうために、 分析 時間を大幅に短縮することができ、 また、 簡易なァフィ二ティ一クロマトグラフ ィ一を利用することで、 簡便 ·迅速且つ高感度に検体を分析することが可能とな る。 また、 ハイブリダィゼ一シヨンや電気泳動の場合に必要な核酸の分離操作を を必要としないため、 試料は粗精製の状態でよく試料の調製も容易である。 さらに、 それぞれの検出しょうとする試料中において、 目的核酸の塩基配列が 微妙に （一塩基以上） 異なる場合も、 ポリメラーゼ等の反応条件を適切に調節す ることにより、 プライマーが目的核酸に完全に相補的である場合とそうでない場 合を区別することができる。 つまり、 ハイブリダィゼーシヨン法などによらない で容易に点突然変異をも検出することができる。  The method for detecting a target nucleic acid according to the present invention can greatly reduce the analysis time by performing an extension reaction using primers without performing a complicated operation in a so-called hybridization reaction. The use of affinity chromatography makes it possible to analyze a sample simply, quickly and with high sensitivity. In addition, since there is no need to perform a nucleic acid separation operation required for hybridization or electrophoresis, the sample is in a roughly purified state and the sample can be easily prepared. Furthermore, even if the base sequence of the target nucleic acid is slightly different (one or more bases) in each sample to be detected, the primers can be completely adjusted to the target nucleic acid by appropriately adjusting the reaction conditions such as polymerase. A distinction can be made between complementary and non-complementary cases. That is, point mutations can be easily detected without using the hybridization method or the like.
また、 それぞれ異なる標識物質で標識された複数のプライマーを使用すれば、 同時に 1種類以上の目的核酸に対するァニ一リング及び伸長反応を行なうことが でき、 同時に複数の目的核酸を検出することも可能である。  In addition, if multiple primers labeled with different labeling substances are used, it is possible to perform annealing and extension reactions on one or more target nucleic acids at the same time, and to detect multiple target nucleic acids simultaneously. It is.

Claims

請求の範囲 The scope of the claims
1. 生物材料中の目的核酸を特異的に検出する方法であって、 1. A method for specifically detecting a target nucleic acid in a biological material,
(a) 目的核酸又はその相補鎖に、 それらのいずれかと相補的な第一標識核酸 プライマ一又は該プライマ一を含むプライマーセットを接触させてプライマー伸 長生成物を形成させる工程、  (a) contacting the target nucleic acid or its complementary strand with a first labeled nucleic acid primer complementary to any of them or a primer set containing the primer to form a primer extension product,
(b) 該第一核酸プライマー伸長生成物を変性させて変性鎖とする工程、 (b) denaturing the first nucleic acid primer extension product into a denatured strand,
(c) 該変性鎖に、 これと相補的な第二標識核酸プライマーを接触させて両標 識プライマー伸長生成物を形成させる工程、 (c) contacting the modified strand with a second labeled nucleic acid primer complementary thereto to form an extension product of both labeled primers,
(d) 該両標識プライマー伸長生成物を、 第一標識核酸プライマー及び第二標 識核酸プライマーの標識と特異的に結合する特異結合試薬を有するクロマトダラ フィー担体に展開させる工程、  (d) developing the two labeled primer extension products on a chromatographic carrier having a specific binding reagent that specifically binds to the labels of the first labeled nucleic acid primer and the second labeled nucleic acid primer;
を行うことを特徵とする目的核酸の検出法。 And a method for detecting a nucleic acid of interest.
2. 工程 （a) と工程 （b) を繰り返し行うものである請求項 1記載の目的核 酸の検出法。  2. The method for detecting a target nucleic acid according to claim 1, wherein the step (a) and the step (b) are repeatedly performed.
3. 生物材料中の目的核酸を特異的に検出するキットであって、  3. A kit for specifically detecting a target nucleic acid in a biological material,
(a) 目的核酸又はその相補鎖に、 それらのいずれかと相補的な第一標識核酸 プライマ一又は該プライマーを含むプライマ一セットを接触させてプライマー伸 長生成物を形成させる手段、  (a) a means for forming a primer extension product by contacting a target nucleic acid or a complementary strand thereof with a first labeled nucleic acid primer complementary to any of them or a set of primers containing the primers;
(b) 該第一核酸プライマー伸長生成物を変性させて変性鎖とする手段、 (b) means for denaturing the first nucleic acid primer extension product into a denatured strand,
(c) 該変性鎖に、 これと相補的な第二標識核酸プライマーを接触させて両標 識プライマー伸長生成物を形成させる手段、 (c) means for contacting the denatured strand with a second labeled nucleic acid primer complementary thereto to form an extension product of both labeled primers,
(d) 該両標識プライマー伸長生成物を、 第一標識核酸プライマー及び第二標 識核酸プライマーの標識と特異的に結合する特異結合試薬を有するクロマトダラ フィ一担体に展開させる手段、  (d) means for developing the both labeled primer extension products on a chromatographic carrier having a specific binding reagent that specifically binds to the labels of the first labeled nucleic acid primer and the second labeled nucleic acid primer,
力、らなる目的核酸の検出キット。 A kit for detecting a target nucleic acid.
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