JP5165177B2 - Method and apparatus for detecting presence or absence of nucleic acid amplification - Google Patents
Method and apparatus for detecting presence or absence of nucleic acid amplification Download PDFInfo
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Description
本発明は、核酸の増幅反応の有無を検出する方法と装置に関する。 The present invention relates to a method and apparatus for detecting the presence or absence of a nucleic acid amplification reaction.
核酸塩基配列の相補性に基づく分析方法は、遺伝的な特徴を直接的に分析することが可能である。そのため、当該分析方法は遺伝的疾患、癌化、微生物の識別等には非常に有力な手段である。また、遺伝子そのものを検出対象とするために、例えば培養のような時間と手間のかかる操作を省略できる場合もある。しかしながら、試料中に存在する目的の核酸量が少ない場合の検出は一般に容易ではなく、標的核酸そのものを、あるいは検出シグナル等を増幅することが必要となる。 Analysis methods based on complementarity of nucleobase sequences can directly analyze genetic characteristics. Therefore, this analysis method is a very effective means for genetic diseases, canceration, identification of microorganisms, and the like. In addition, since the gene itself is a detection target, a time-consuming and laborious operation such as culture may be omitted. However, detection when the amount of the target nucleic acid present in the sample is small is generally not easy, and it is necessary to amplify the target nucleic acid itself or a detection signal.
核酸の増幅産物を検出する最も一般的な方法は、増幅反応後の溶液をアガロースゲル電気泳動にアプライし、エチジウムブロマイド等の蛍光インターカレーターを結合させて特異的な蛍光を観察するというものである。ここで、他のＤＮＡが混在するおそれがなく、増幅産物の有無のみを知りたい場合には、電気泳動を省略して増幅反応後の溶液に蛍光インターカレーターを加えて蛍光を観察することも可能である。ただし、これらの方法は簡便であるものの、蛍光を観察するためのＵＶランプと暗室が必要である。 The most common method for detecting nucleic acid amplification products is to apply a solution after amplification reaction to agarose gel electrophoresis and to observe a specific fluorescence by binding a fluorescent intercalator such as ethidium bromide. . Here, if there is no risk of mixing with other DNA and you only want to know the presence or absence of amplification products, you can omit the electrophoresis and add fluorescence intercalator to the solution after amplification reaction to observe the fluorescence. It is. However, although these methods are simple, a UV lamp and a darkroom for observing fluorescence are required.
固相等の支持体上で核酸を発色させる方法には、酵素を利用し金属−硫黄錯体を固着する方法(例えば、特許文献１参照）、金属染色法で染色した核酸に色素前駆体と補力剤を作用させる方法(例えば、特許文献２参照）があるが、いずれも煩雑な工程を必要とする。 As a method of coloring a nucleic acid on a support such as a solid phase, a method of fixing a metal-sulfur complex using an enzyme (see, for example, Patent Document 1), a nucleic acid stained by a metal staining method and a dye precursor and a complement. Although there is a method (for example, refer to Patent Document 2) in which a force agent is applied, all of them require complicated steps.
蛍光色素をはじめとする各種標識物質で標識したプライマーやヌクレオチドを用いて増幅反応を行い、増幅産物に取り込まれた標識を観察する方法もあるが(例えば、特許文献３参照）、増幅産物に取り込まれなかったフリーの標識プライマー（あるいはヌクレオチド）を分離する操作が必要であり、反応液量が微量である核酸増幅反応には適さない。また、標識プライマーやヌクレオチドは高価である。 There is also a method of performing an amplification reaction using primers and nucleotides labeled with various labeling substances including fluorescent dyes and observing the label incorporated into the amplification product (for example, see Patent Document 3), but incorporated into the amplification product. An operation for separating the free labeled primer (or nucleotide) that has not been performed is necessary, and it is not suitable for a nucleic acid amplification reaction in which the amount of the reaction solution is very small. Also, labeled primers and nucleotides are expensive.
電気泳動ゲルや膜等の支持体および標識物質を使用しない検出法には、核酸増幅反応液に偏光を通過させその偏光の旋光度または円偏光二色性を測定する方法(例えば、特許文献４参照）、伸長した増幅産物の偏光成分の変化を検知する方法(例えば、特許文献５、６および７参照）がある。また、増幅反応に伴い生成するピロリン酸とマグネシウムによる不溶性物質の沈殿を観察する方法(例えば、特許文献８参照）も開発されているが、沈殿による検出は極めて簡便で実用的であるものの、シグナルとしての認識性および訴求力にやや乏しい面もある。さらに、ピロリン酸を対象とする分析方法として、ピロリン酸に対して、酸化酵素を含む酵素反応試薬で処理し、酸化酵素が作用する際に起こる電子移動を、電気化学的活性インターカレータの存在下で増幅し、電気化学的に電流として検出する方法もあるが操作の煩雑さは否めない(例えば、特許文献９参照）。いずれにせよ、上述した方法には、増幅反応液の色調変化を簡単に観察できる極めて簡便で明確な核酸増幅の有無の検出方法についての記載はない。 A detection method that does not use a support such as an electrophoresis gel or a membrane and a labeling substance is a method in which polarized light is passed through a nucleic acid amplification reaction solution and the optical rotation or circular dichroism of the polarized light is measured (for example, Patent Document 4). (See, for example, Patent Documents 5, 6 and 7). In addition, a method for observing precipitation of insoluble substances due to pyrophosphate and magnesium generated in the amplification reaction (see, for example, Patent Document 8) has been developed, but detection by precipitation is extremely simple and practical. There is also a slight lack of recognition and appeal. Furthermore, as an analysis method for pyrophosphate, the electron transfer that occurs when pyrophosphate is treated with an enzyme reaction reagent containing an oxidase and the oxidase acts is detected in the presence of an electrochemically active intercalator. There is a method in which the current is amplified and electrochemically detected as a current, but the operation is undeniable (see, for example, Patent Document 9). In any case, the above-described method does not describe a very simple and clear method for detecting the presence or absence of nucleic acid amplification that allows easy observation of the color change of the amplification reaction solution.
本発明は、核酸の増幅反応の有無または増幅過程を、複雑な操作や特殊な装置を必要とせずに、簡便かつ迅速に検出できる方法を提供することを目的とする。 An object of the present invention is to provide a method capable of easily and rapidly detecting the presence or absence of an amplification reaction of a nucleic acid or an amplification process without requiring a complicated operation or a special apparatus.
本発明者は、上記課題を解決すべく鋭意研究を行った結果、核酸の増幅反応において、
反応生成物と結合する金属イオンと、核酸とは結合しない金属指示薬との結合能を指標とした高感度な核酸増幅の有無の検出法と、核酸の増幅過程をモニタリングする方法ならびに試薬キット、さらには核酸増幅の有無を検出する簡易装置を見出し、本発明を完成させた。
As a result of intensive studies to solve the above problems, the present inventor, in nucleic acid amplification reaction,
A highly sensitive method for detecting the presence or absence of nucleic acid amplification based on the binding ability of a metal ion that binds to a reaction product and a metal indicator that does not bind to a nucleic acid, a method and a reagent kit for monitoring the nucleic acid amplification process, and Found a simple apparatus for detecting the presence or absence of nucleic acid amplification, and completed the present invention.
すなわち、本発明は、核酸の合成または増幅反応における、ｄＮＴＰｓとピロリン酸の結合能の差に基づく反応液中の金属イオン量の変化を、金属指示薬によって検知する核酸合成または増幅の有無の検出方法である。さらに、本発明は、ポリヌクレオチド鎖上の標的領域を増幅し、増幅反応に伴い変化する反応液中の金属イオンを指示薬により呈色させ、核酸の合成または増幅の有無すなわち標的核酸の存在を簡便に検出する方法と、核酸の増幅過程をモニターする方法ならびに試薬キット、さらには核酸増幅の有無を検出する簡易装置に関するものであり、以下の構成からなる。
（１）核酸の増幅反応において、反応生成物に結合する金属イオンと、核酸とは結合しない化合物との結合による呈色および／または蛍光を指標とすることを特徴とする、核酸増幅の有無を検出する方法。
（２）さらに、反応生成物と金属イオンとの結合によって生じる不溶性物質による濁度または沈殿を指標とすることを特徴とする、（１）記載の方法。
（３）核酸の増幅反応において、反応生成物に結合する金属イオンと金属指示薬との呈色および／または蛍光反応を指標とすることを特徴とする、核酸増幅の有無を検出する方法。
（４）金属イオンが、Ｍｇ（II）、Ｍｎ（II）、Ｎｉ（II）、Ｆｅ（II）およびＺｎ（II）から選択される少なくとも１種である（１）〜（３）記載の方法。
（５）金属指示薬が、エリオクロムブラックＴ、ヒドロキシナフトールブルー、チモールフタレインコンプレクソン、メチルチモールブルー、キシリジルブルー−１、クロロホスフォナゾ−３およびカルセインから選択される少なくとも１種である（３）記載の方法。
（６）反応液中の金属指示薬の濃度が、０．０５〜１０００μＭの範囲である（５）記載の方法。
（７）核酸の増幅反応がＬＡＭＰ法であることを特徴とする（１）〜（６）記載の核酸増幅の有無を検出する方法。
（８）反応液の入った容器を反応温度が一定に保てるサンプルホルダーに設置し、反応液に光を照射して、濁度、呈色または蛍光を検出することを特徴とする（７）記載の方法。
（９）（７）記載の核酸増幅の有無を濁度、呈色または蛍光によって検出する装置であって、
（ａ）反応温度を一定に保てる保温機能を有するサンプルホルダー、
（ｂ）前記（ａ）に挿入した反応容器の中の反応液に、光が照射できるように配置された光源、ならびに
（ｃ）反応液の濁度、呈色または蛍光を検知できるように配置された検出器、含む装置。
（１０）光源を切り換えることにより濁度、呈色または蛍光による検出を行えるようにしたことを特徴とする（９）記載の装置。
（１１）核酸を増幅するために必要な試薬に加え、金属指示薬を含む試薬キット。
（１２）金属指示薬が、エリオクロムブラックＴ、ヒドロキシナフトールブルー、チモールフタレインコンプレクソン、メチルチモールブルー、キシリジルブルー−１、クロロホスフォナゾ−３およびカルセインから選択される少なくとも１種である（１１）記載のキット。
That is, the present invention relates to a method for detecting the presence or absence of nucleic acid synthesis or amplification by detecting a change in the amount of metal ions in a reaction solution based on the difference in binding ability between dNTPs and pyrophosphate in a nucleic acid synthesis or amplification reaction using a metal indicator. It is. Furthermore, the present invention amplifies the target region on the polynucleotide chain, and colors the metal ion in the reaction solution that changes with the amplification reaction with the indicator, so that the presence or absence of the nucleic acid synthesis or amplification, that is, the presence of the target nucleic acid can be simplified. The present invention relates to a detection method, a method for monitoring a nucleic acid amplification process, a reagent kit, and a simple apparatus for detecting the presence or absence of nucleic acid amplification, and has the following configuration.
(1) In nucleic acid amplification reaction, the presence or absence of nucleic acid amplification, characterized by coloration and / or fluorescence resulting from the binding of a metal ion that binds to a reaction product and a compound that does not bind to nucleic acid How to detect.
(2) The method according to (1), further characterized by using as an index turbidity or precipitation due to an insoluble substance generated by the combination of the reaction product and metal ions.
(3) A method for detecting the presence or absence of nucleic acid amplification, characterized in that, in an amplification reaction of nucleic acid, coloration and / or fluorescence reaction between a metal ion binding to a reaction product and a metal indicator is used as an index.
(4) The method according to (1) to (3), wherein the metal ion is at least one selected from Mg (II), Mn (II), Ni (II), Fe (II) and Zn (II) .
(5) The metal indicator is at least one selected from Eriochrome Black T, Hydroxynaphthol Blue, Thymolphthalein Complexone, Methylthymol Blue, Xylidyl Blue-1, Chlorophosphonazo-3 and Calcein ( 3) The method as described.
(6) The method according to (5), wherein the concentration of the metal indicator in the reaction solution is in the range of 0.05 to 1000 μM.
(7) The method for detecting the presence or absence of nucleic acid amplification according to (1) to (6), wherein the nucleic acid amplification reaction is a LAMP method.
(8) The container containing the reaction solution is placed in a sample holder capable of keeping the reaction temperature constant, and the reaction solution is irradiated with light to detect turbidity, coloration, or fluorescence. the method of.
(9) An apparatus for detecting the presence or absence of nucleic acid amplification according to (7) by turbidity, coloration or fluorescence,
(A) a sample holder having a heat retaining function capable of keeping the reaction temperature constant;
(B) A light source arranged so that the reaction liquid in the reaction vessel inserted in (a) can be irradiated with light, and (c) arranged so that turbidity, coloration or fluorescence of the reaction liquid can be detected. Detector, including device.
(10) The apparatus according to (9), wherein detection by turbidity, coloration or fluorescence can be performed by switching a light source.
(11) A reagent kit containing a metal indicator in addition to reagents necessary for amplifying nucleic acid.
(12) The metal indicator is at least one selected from Eriochrome Black T, Hydroxynaphthol Blue, Thymolphthalein Complexone, Methylthymol Blue, Xylidyl Blue-1, Chlorophosphonazo-3 and Calcein ( 11) The kit according to the description.
本発明の方法により、試料中に存在する目的の標的核酸の増幅の有無を簡単に検出することができる。
以下、本発明について更に詳細に説明する。
By the method of the present invention, it is possible to easily detect the presence or absence of amplification of a target nucleic acid of interest in a sample.
Hereinafter, the present invention will be described in more detail.
本発明は、核酸またはポリヌクレオチド鎖上の標的領域を合成または増幅した後もしくは合成または増幅中に、合成反応または増幅反応に伴い変化する反応液中の金属イオンと金属指示薬との呈色または蛍光反応により、核酸の合成または増幅反応を検出するものである。反応によって生じる色調の変化は、肉眼でも観察可能であるため、増幅反応の有無を、複雑な操作や特殊な装置を必要とせずに、簡便かつ迅速に検出することを特徴としている。 The present invention relates to the coloration or fluorescence of a metal ion and a metal indicator in a reaction solution that changes with a synthesis reaction or an amplification reaction after or during synthesis or amplification of a target region on a nucleic acid or polynucleotide chain. The reaction detects nucleic acid synthesis or amplification reaction. Since the change in color tone caused by the reaction can be observed with the naked eye, it is characterized in that the presence or absence of the amplification reaction can be detected easily and quickly without requiring a complicated operation or a special device.
１．検出対象
本発明において、検出対象となる金属イオンは、核酸増幅反応の結果生じるピロリン酸と結合可能な金属イオンである。一般にタリウム（I）以外のアルカリ金属およびアルカリ土金属の多くはピロリン酸と結合して水に不溶、あるいは難溶性の塩を形成するため、好適に使用できる。増幅反応液中には、通常、ポリメラーゼの活性化剤であるマグネシウムイオンが十分量存在しているため、これをそのまま指標としてもよい。また、別途マグネシウムイオンと異なる金属イオンを加えることにより、マグネシウムイオンとピロリン酸の結合が抑制され、ポリメラーゼ活性を低下させない効果が期待できる。この方法では、マグネシウムのように過剰量添加する必要がないため、金属指示薬の選択範囲が広がる。しかしながら、マグネシウムと同じアルカリ土類金属であるカルシウムは、ポリメラーゼ活性を阻害するため使用に適さない。マグネシウム以外に好適な金属イオンとして、Ｍｎ（II）、Ｎｉ（II）、Ｆｅ（II）、Ｚｎ（II）等が挙げられる。
1. In the detection subject invention, metal ions to be detected is capable of binding metal ions resulting pyrophosphate nucleic acid amplification reaction. In general, many of alkali metals and alkaline earth metals other than thallium (I) are combined with pyrophosphoric acid to form a salt that is insoluble or hardly soluble in water, and thus can be suitably used. Usually, a sufficient amount of magnesium ion, which is an activator of polymerase, is present in the amplification reaction solution, and this may be used as an index as it is. In addition, by adding a metal ion different from magnesium ion, the binding between magnesium ion and pyrophosphate can be suppressed, and the effect of not lowering the polymerase activity can be expected. In this method, since it is not necessary to add an excessive amount like magnesium, the selection range of the metal indicator is expanded. However, calcium, the same alkaline earth metal as magnesium, is not suitable for use because it inhibits polymerase activity. Examples of suitable metal ions other than magnesium include Mn (II), Ni (II), Fe (II), Zn (II), and the like.
ここで、マグネシウム以外に、別途金属イオンを添加する場合のその濃度は、０．０１〜１００ｍＭの範囲であることが好ましく、より好ましくは、０．０５〜２０ｍＭの範囲である。濃度が０．０１ｍＭより低いときは、金属指示薬によっては呈色が十分でなく、１００ｍＭより高い濃度では、ポリメラーゼ活性の低下により、増幅反応に影響を及ぼすときがある。 Here, in addition to magnesium, the concentration in the case of adding a metal ion separately is preferably in the range of 0.01 to 100 mM, more preferably in the range of 0.05 to 20 mM. When the concentration is lower than 0.01 mM, coloration is not sufficient depending on the metal indicator, and when the concentration is higher than 100 mM, the amplification reaction may be affected due to a decrease in polymerase activity.
前記金属イオンの検出は、水溶性金属指示薬によるのが好ましい。本発明では、増幅に伴い減少する反応液中の金属イオンを検出するため、金属イオン濃度に比例して発色を呈する指示薬ではなく、金属イオンと金属指示薬とのキレート化によって吸光スペクトルが変化し、その変化を観察できるものの方が好ましい。例えば、エリオクロムブラックＴ（ＢＴ）はｐＨ１０において種々の金属の共存により赤色を呈し、非存在下では青色を呈するので好適である。一方、この色調の変化を利用して増幅反応をモニタリングするためには、反応液のｐＨ、反応温度（６０℃以上）、ポリメラーゼに対する阻害等を考慮しなければならない。また、マグネシウム以外の金属イオンを指標とする場合は、共存するマグネシウムの影響も考慮する必要がある。このような条件を満たす金属指示薬として、ＢＴの他、ヒドロキシナフトールブルー（ＨＮＢ）、チモールフタレインコンプレクソン（ＴＰＣ）、メチルチモールブルー（ＭＴＢ）、キシリジルブルー1（ＸＢ−１ ）、クロロホスフォナゾ−３（Ｃｈ−３）およびカルセインが挙げられる。これらの指示薬は、単独または組み合わせて使用できる。 The detection of the metal ions is preferably performed by a water-soluble metal indicator. In the present invention, in order to detect the metal ion in the reaction solution that decreases with amplification, instead of an indicator that develops color in proportion to the metal ion concentration, the absorption spectrum changes due to chelation of the metal ion and the metal indicator, Those that can observe the change are preferred. For example, Eriochrome Black T (BT) is suitable because it exhibits a red color due to the coexistence of various metals at pH 10 and a blue color in the absence. On the other hand, in order to monitor the amplification reaction using this change in color tone, the pH of the reaction solution, the reaction temperature (60 ° C. or higher), the inhibition of the polymerase, etc. must be taken into consideration. Moreover, when using metal ions other than magnesium as an index, it is necessary to consider the influence of coexisting magnesium. As a metal indicator satisfying such conditions, in addition to BT, hydroxynaphthol blue (HNB), thymolphthalein complexone (TPC), methylthymol blue (MTB), xylidyl blue 1 (XB-1), chlorophospho Nazo-3 (Ch-3) and calcein are mentioned. These indicators can be used alone or in combination.
金属指示薬の濃度は、０．０５〜１０００μＭの範囲であることが好ましく、より好ましくは、０．５〜５００μＭの範囲である。濃度が０．０５μＭより低いときは、金属イオンによっては呈色が十分でなく、１０００μＭより高い濃度では、反応液のバックグランドが上昇し、呈色による増幅の有無が確認しにくくなる。 The concentration of the metal indicator is preferably in the range of 0.05 to 1000 μM, more preferably in the range of 0.5 to 500 μM. When the concentration is lower than 0.05 μM, the coloration is not sufficient depending on the metal ions, and when the concentration is higher than 1000 μM, the background of the reaction solution increases and it is difficult to confirm the presence or absence of amplification due to the coloring.
ここで、本発明においてポリヌクレオチドとは、増幅の対象となる核酸を意味し、一般的にはＤＮＡとＲＮＡの双方を含む。核酸は、一般に生物学的試料に含まれる。生物学的試料とは、動物、植物または微生物の組織、細胞、培養物若しくは排泄物、またはそれらの抽出物を意味する。また、生物学的試料には、ウイルスやマイコプラズマのような細胞内寄生体のゲノムＤＮＡ、あるいはＲＮＡが含まれる。また核酸は、前記生物学的試料に含まれる核酸から誘導されたものであってもよい。たとえば、ｍＲＮＡをもとに合成されたｃＤＮＡや、生物学的試料に由来する核酸をもとに増幅された核酸等が挙げられる。 Here, in the present invention, the polynucleotide means a nucleic acid to be amplified, and generally includes both DNA and RNA. Nucleic acids are generally contained in biological samples. A biological sample means an animal, plant or microbial tissue, cell, culture or excrement, or an extract thereof. Biological samples also include genomic DNA or RNA of intracellular parasites such as viruses and mycoplasmas. The nucleic acid may be derived from a nucleic acid contained in the biological sample. Examples thereof include cDNA synthesized based on mRNA and nucleic acid amplified based on nucleic acid derived from a biological sample.
２．核酸の合成反応
核酸の合成反応では、使用する酵素により核酸の末端にヌクレオチドを付加する過程でピロリン酸が生成する場合がある。本発明は、このような酵素による核酸の合成反応で生成されたピロリン酸と金属イオンの結合能を指標として核酸の存在を検出することができる。前記酵素としては、特に限定されるものではなく、Ｅ．Ｃｏｌｉ ＤＮＡ ポリメラーゼ、Ｔａｑ ＤＮＡ ポリメラーゼ、Ｔ４ ＤＮＡ ポリメラーゼ、逆転写酵素（ Reverse Transcriptase ）、ＳＰ６ ＲＮＡ ポリメラーゼ、Ｔ７ ＲＮＡ ポリメラーゼ、ターミナルデオキシヌクレオチジルトランスフェラーゼ、Ｐｏｌｙ（Ａ）ポリメラーゼ、Ｂｓｔ ＤＮＡポリメラーゼ、Ｖｅｎｔ ＤＮＡポリメラーゼ等が挙げられ、各酵素の反応は、公知の任意の条件で行うことができる（ T. Maniatis et al., Molecular cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 )。
2. Nucleic Acid Synthesis Reaction In a nucleic acid synthesis reaction, pyrophosphate may be generated in the process of adding nucleotides to the end of the nucleic acid by the enzyme used. The present invention can detect the presence of a nucleic acid using as an index the binding ability of pyrophosphate and metal ions produced by a nucleic acid synthesis reaction by such an enzyme. The enzyme is not particularly limited. Examples include Coli DNA polymerase, Taq DNA polymerase, T4 DNA polymerase, Reverse Transcriptase, SP6 RNA polymerase, T7 RNA polymerase, terminal deoxynucleotidyl transferase, Poly (A) polymerase, Bst DNA polymerase, Vent DNA polymerase and the like. The reaction of each enzyme can be performed under any known conditions (T. Maniatis et al., Molecular cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989).
３．増幅反応
本発明の検出対象は、ピロリン酸と結合する金属イオンであることから、本発明において適用される増幅反応は、ヌクレオチドが核酸鎖に取り込まれる際にピロリン酸が生成する反応であれば、特に限定されるものではない。例えば、in vitroにおける核酸の増幅技術として現在最も一般的な方法であるＰＣＲ（ Polymerase Chain Reaction ）法の他、ＬＡＭＰ（ Loop-Mediated Isothermal Amplification ）法と呼ばれる増幅法（特許第３３１３３５８号等）、ＳＤＡ（ Strand Displacement Amplification ）法（特公平７−１１４７１８号公報等)、ＮＡＳＢＡ( Nucleic Acid Sequence Based Amplification ）法（特許第２６５０１５９号）等に適用することができる。
3. Amplification reaction Since the detection target of the present invention is a metal ion that binds to pyrophosphate, the amplification reaction applied in the present invention is a reaction that generates pyrophosphate when nucleotides are incorporated into a nucleic acid chain. It is not particularly limited. For example, in addition to the PCR (polymerase chain reaction) method, which is the most common method for nucleic acid amplification in vitro, an amplification method called a LAMP (loop-Mediated Isothermal Amplification) method (Patent No. 3313358, etc.), SDA It can be applied to the (Strand Displacement Amplification) method (Japanese Patent Publication No. 7-114718), the NASBA (Nucleic Acid Sequence Based Amplification) method (Japanese Patent No. 2650159), and the like.
前期増幅反応のうちＬＡＭＰ法は、増幅対象となる塩基配列の末端にループ構造を形成し、そこを起点としてポリメラーゼによる伸長反応が起きると同時に、ループ内の領域にハイブリダイズしたプライマーが、鎖置換反応により核酸鎖を伸長しながら先の伸長反応の産物を１本鎖に解離させていくというものである。生成した１本鎖核酸はその末端に自己相補性領域を持つため、末端にループを形成し新たな伸長反応が始まる。実際のＬＡＭＰ法では等温で進行するため、上に述べた反応は同時に並行して起こる。ＬＡＭＰ法の特徴としては、等温で進行する鎖置換型の反応であることの他に、増幅産物の量が非常に多いことが挙げられる。これは、ポリメラーゼが失活する原因である熱変性の操作が含まれていないことも一因である。増幅産物の量が多いということは、生成するピロリン酸の量すなわちピロリン酸と反応する金属イオンの量も多いため、本発明を適用する核酸増幅法としてＬＡＭＰ法は好適である。 Among the previous amplification reactions, the LAMP method forms a loop structure at the end of the base sequence to be amplified, and at the same time, an extension reaction by polymerase occurs. At the same time, the primer hybridized to the region in the loop replaces the strand. The product of the previous extension reaction is dissociated into a single strand while the nucleic acid strand is extended by the reaction. Since the generated single-stranded nucleic acid has a self-complementary region at its end, a loop is formed at the end and a new extension reaction starts. Since the actual LAMP method proceeds isothermally, the reactions described above occur simultaneously in parallel. As a feature of the LAMP method, in addition to the strand displacement reaction that proceeds isothermally, the amount of amplification product is very large. This is partly due to the fact that the thermal denaturation operation, which causes the polymerase to be inactivated, is not included. Since the amount of amplification product is large, the amount of pyrophosphoric acid produced, that is, the amount of metal ions that react with pyrophosphoric acid is large, so the LAMP method is suitable as a nucleic acid amplification method to which the present invention is applied.
ここで、ＬＡＭＰ法において使用されるオリゴヌクレオチドプライマーは、標的核酸の塩基配列の計６領域から設計・作製された、ループ構造の形成に関与する２種類の内部プライマー（ＦＡプライマー、ＲＡプライマー）と、２種類の外部プライマー（Ｆ３プライマー、Ｒ３プライマー）の、少なくとも計４種類のオリゴヌクレオチドプライマーが挙げられる。 Here, the oligonucleotide primers used in the LAMP method are designed and produced from a total of 6 regions of the base sequence of the target nucleic acid, and two types of internal primers (FA primer and RA primer) involved in the formation of the loop structure. There are at least four types of oligonucleotide primers of two types of external primers (F3 primer, R3 primer).
４．検出
本発明においては、反応液中の金属イオンを金属指示薬によって検出する。検出操作の違いにより、以下の通り、定性的または定量的に検出することが可能である。検出対象である金属イオンは、増幅反応に用いられたヌクレオチドから生成したピロリン酸と結合して不溶性物質を形成し、反応液中の金属イオンは増幅反応の進行に伴い減少する。
4). Detection In the present invention, metal ions in the reaction solution are detected with a metal indicator. Depending on the detection operation, it is possible to detect qualitatively or quantitatively as follows. The metal ion to be detected binds to pyrophosphate generated from the nucleotide used in the amplification reaction to form an insoluble substance, and the metal ion in the reaction solution decreases as the amplification reaction proceeds.
（１）目視による検出
増幅反応により減少する反応液中の金属イオンを検出する最も簡便な方法は、透明な容器の横からあるいは不透明容器の上部から、金属指示薬による呈色または蛍光を目視により観察することである。
（２）吸光度による測定
反応液の吸光度を測定することにより、増幅産物を検出することもできる。吸光度の測定には、汎用の分光光度計等を用いることができる。測定波長は、金属イオンおよび金属指示薬の種類によって適宜設定すればよい。また、吸光度測定により、特定のサイクル数または一定時間で反応を停止した反応液を用いて、試料中の核酸を定量することも可能である。さらに本発明においては、特に吸光度を経時的に測定することで、反応時間の経過と共に反応産物がどのように推移したか、そのモニタリングが可能となる。
（３）蛍光による測定
金属指示薬カルセインは、呈色と同時に蛍光も発するため蛍光の観察により増幅反応の有無を検出することもできる。また増幅反応がネガティブで青色、ポジティブで無色となるＴＰＣにさらに一般的な蛍光色素（フルオレセイン等）を添加すれば、ポジティブにおいても蛍光が観察されるようになる。このように、適当な蛍光色素を選択して添加することで、より広範な波長域で増幅反応の有無の検出またはモニタリングが可能となる。
(1) Visual detection The simplest method for detecting metal ions in a reaction solution that decreases due to an amplification reaction is to visually observe coloration or fluorescence by a metal indicator from the side of a transparent container or from the top of an opaque container. It is to be.
(2) Measurement by absorbance The amplification product can also be detected by measuring the absorbance of the reaction solution. A general-purpose spectrophotometer or the like can be used for measuring the absorbance. What is necessary is just to set a measurement wavelength suitably with the kind of metal ion and metal indicator. It is also possible to quantify the nucleic acid in the sample by measuring the absorbance using a reaction solution that has stopped the reaction at a specific number of cycles or a certain time. Furthermore, in the present invention, it is possible to monitor how the reaction product has changed over time by measuring the absorbance over time.
(3) Measurement by fluorescence Since the metal indicator calcein emits fluorescence simultaneously with coloration, the presence or absence of an amplification reaction can also be detected by observation of fluorescence. In addition, if a more general fluorescent dye (fluorescein or the like) is added to TPC in which the amplification reaction is negative and blue, and positive and colorless, fluorescence can be observed even in positive. Thus, by selecting and adding an appropriate fluorescent dye, it is possible to detect or monitor the presence or absence of an amplification reaction in a wider wavelength range.
例えば、前記蛍光を利用したモニタリングに関しては、以下の構成を備えた簡易装置を用いて実施できる。すなわち、核酸増幅の反応温度を一定に保てる保温機能を備えたサンプルホルダーと、前記サンプルホルダーの、例えば下部に設けた開放口からサンプルホルダーに導入された反応容器の中の反応液に励起光が照射できるように配置された光源と、反応液から放射された蛍光を、光軸に対して９０°の方向での検知できるように配置された検出器とを備えた装置である。さらに、前記検出器に、所要の波長のみを通過させるためのフィルターを兼ね備えていてもよい。 For example, the monitoring using the fluorescence can be performed using a simple device having the following configuration. That is, a sample holder having a heat retaining function capable of keeping the reaction temperature of nucleic acid amplification constant, and excitation light is applied to the reaction liquid in the reaction container introduced into the sample holder from an opening provided in the lower part of the sample holder, for example. It is an apparatus comprising a light source arranged so as to be able to irradiate and a detector arranged so as to detect fluorescence emitted from the reaction solution in a direction at 90 ° with respect to the optical axis. Furthermore, the detector may also have a filter for allowing only a required wavelength to pass therethrough.
ここで、本発明で用いるサンプルホルダーは、反応容器の導入口、光の通過する開放口を有しており、核酸増幅中の反応液の温度が一定に保たれれば特に限定されない。そして、前記光源は、紫外線（ＵＶ）ランプ、発光ダイオード（ＬＥＤ）、ケミカルライト、レーザー光等から選択され、吸光度または蛍光等の検出手段により、切り換えができるように配置されていてもよい。前記検出器は、ＣＣＤ、ＣＭＯＳ、フォトダイード等から選択される。そして、所要の波長のみを通過させるためのフィルターとは、反応液から放射される固有の波長の帯域光のみを透過させ、それ以外の波長域を遮断する目的のものである。 Here, the sample holder used in the present invention has an inlet for the reaction vessel and an opening through which light passes, and is not particularly limited as long as the temperature of the reaction solution during nucleic acid amplification is kept constant. The light source may be selected from an ultraviolet (UV) lamp, a light emitting diode (LED), a chemical light, a laser beam, or the like, and may be arranged so that it can be switched by a detection means such as absorbance or fluorescence. The detector is selected from CCD, CMOS, photo diode and the like. The filter for allowing only the required wavelength to pass is for the purpose of transmitting only the band light of the specific wavelength emitted from the reaction solution and blocking the other wavelength ranges.
以下、実施例により本発明をさらに具体的に説明する。ただし、本発明は、これら実施例において、その技術的範囲が限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited in these examples.
実施例１：ＬＡＭＰ反応による核酸増幅の金属指示薬による検出
〔反応液組成〕
ＬＡＭＰ用反応液組成(２５μＬ中)
２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．８）
１０ｍＭ ＫＣｌ
１０ｍＭ （ＮＨ4）2ＳＯ4
８ｍＭ ＭgＳＯ4
０．１％ Ｔｗｅｅｎ２０
０．８Ｍ Ｂｅｔａｉｎ
１．４ｍＭ ｄＮＴＰｓ
８Ｕ ＢｓｔＤＮＡポリメラーゼ( New England Biolab社製 )
１．６μＭ ＦＡプライマー
１．６μＭ ＲＡプライマー
０．４μＭ Ｆ３プライマー
０．４μＭ Ｒ３プライマー
Example 1: Detection of nucleic acid amplification by LAMP reaction with a metal indicator [reaction solution composition]
Reaction solution composition for LAMP (in 25 μL)
20 mM Tris-HCl (pH 8.8)
10 mM KCl
10 mM (NH 4 ) 2 SO 4
8 mM MgSO 4
0.1% Tween20
0.8M Betaine
1.4 mM dNTPs
8U Bst DNA polymerase (New England Biolab)
1.6 μM FA primer 1.6 μM RA primer 0.4 μM F3 primer 0.4 μM R3 primer
前記ＬＡＭＰ用反応液に、ＬＡＭＰ反応の鋳型としてＰＳＡ(前立腺特異抗原)ＤＮＡを６×１０-20Ｍ、金属指示薬としてエリオクロムブラックＴ（ＢＴ）、ヒドロキシナフトールブルー（ＨＮＢ）、チモールフタレインコンプレクソン（ＴＰＣ）およびメチルチモールブルー（ＭＴＢ）をそれぞれ５０μＭ、キシリジルブルー−１（ＸＢ−１）は１００μＭの濃度となるように添加し、増幅反応を６５℃で６０分を行った。ＴＰＣ、ＸＢ−１は水に難溶のため、予め０．０２ＭのＮａＯＨに溶解した１０ｍＭ溶液を添加した。なお、鋳型ＤＮＡを添加した反応液をポジティブ（＋）、鋳型ＤＮＡを添加しなかった反応液をネガティブ（−）（以下それぞれ「ポジティブ」「ネガティブ」）とした。本増幅反応においては、鋳型中に含まれる以下の配列(配列番号１)を目的のポリヌクレオチドとした。
5'-TGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCCTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCACACACCC-3'(配列番号１)
In the reaction solution for LAMP, 6 × 10 −20 M of PSA (prostate specific antigen) DNA as a template for LAMP reaction, Eriochrome black T (BT), hydroxynaphthol blue (HNB), thymolphthalein complexone as metal indicator (TPC) and methylthymol blue (MTB) were added to a concentration of 50 μM and xylidyl blue-1 (XB-1) to a concentration of 100 μM, respectively, and the amplification reaction was performed at 65 ° C. for 60 minutes. Since TPC and XB-1 are hardly soluble in water, a 10 mM solution previously dissolved in 0.02 M NaOH was added. The reaction solution to which the template DNA was added was positive (+), and the reaction solution to which the template DNA was not added was negative (−) (hereinafter “positive” and “negative”, respectively). In this amplification reaction, the following polynucleotide (SEQ ID NO: 1) contained in the template was used as the target polynucleotide.
5'-TGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCCTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCACACACCC-3 '(SEQ ID NO: 1
また、反応液組成中のＦＡプライマー、ＲＡプライマーならびにＦ３、Ｒ３プライマーは、配列番号１に示す塩基配列に基づいて以下のように設計した。
・ＦＡプライマー
5'-TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG-3'(配列番号２)
・ＲＡプライマー
5'-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG-3'(配列番号３)
・Ｆ３プライマー
5'-TGCTTGTGGCCTCTCGTG-3'(配列番号４)
・Ｒ３プライマー
5'-GGGTGTGTGAAGCTGTG-3'(配列番号５)
The FA primer, RA primer and F3 and R3 primers in the reaction solution composition were designed as follows based on the base sequence shown in SEQ ID NO: 1.
・ FA primer
5'-TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG-3 '(SEQ ID NO: 2)
・ RA primer
5'-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG-3 '(SEQ ID NO: 3)
・ F3 primer
5'-TGCTTGTGGCCTCTCGTG-3 '(SEQ ID NO: 4)
・ R3 primer
5'-GGGTGTGTGAAGCTGTG-3 '(SEQ ID NO: 5)
増幅後の反応液の色調を目視により観察した。また、反応液のスペクトルおよび吸光度の測定に関しては、遠心操作によって反応液中のピロリン酸マグネシウムの沈殿物を除去した後、上清８０μＬを、 分光光度計Ｕｌｔｏｒｏｓｐｅｃ２０００（ Pharmacia社製 ）を用いて、波長３００〜８００ｎｍの吸光スペクトルと、可視領域内での変化の大きい極大吸収波長における吸光度を測定した。 The color tone of the reaction solution after amplification was visually observed. Regarding the measurement of the spectrum and absorbance of the reaction solution, after removing the magnesium pyrophosphate precipitate in the reaction solution by centrifugation, 80 μL of the supernatant was analyzed using a spectrophotometer Ultrospec 2000 (manufactured by Pharmacia). The absorbance spectrum at 300 to 800 nm and the absorbance at the maximum absorption wavelength having a large change in the visible region were measured.
その結果を図１〜図５および以下に示す。すべての指示薬について概ね５５０〜７００ｎｍの１つの領域にスペクトル変化が認められた。また、遠心後の反応液の色調差も顕著であり、肉眼で容易に増幅の有無を確認することができた。
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指示薬 鋳型添加 色調 吸光度（極大吸収波長）
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ＢＴ − 赤紫 ０．２４７（６５０ｎｍ）
＋ 青 ０．６２９（ 同 上 ）
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ＨＮＢ − 青紫 ０．５７９（６５０ｎｍ）
＋ 青 １．１１９（ 同 上 ）
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ＴＰＣ − 青 ０．５７３（６００ｎｍ）
＋ 無色 ０．１８５（ 同 上 ）
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ＭＴＢ − 淡青 １．１５３（６１０ｎｍ）
＋ 濃青 ０．７００（ 同 上 ）
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ＸＢ−１ − 橙 ０．１７８（６００ｎｍ）
＋ 桃 ０．４４４（ 同 上 ）
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The results are shown in FIGS. Spectral changes were observed in one region of approximately 550 to 700 nm for all indicators. In addition, the color difference of the reaction solution after centrifugation was remarkable, and the presence or absence of amplification could be easily confirmed with the naked eye.
-------------------------------------------------- ------------------
Indicator Template addition Color tone Absorbance (Maximum absorption wavelength)
-------------------------------------------------- ------------------
BT-Magenta 0.247 (650nm)
+ Blue 0.629 (same as above)
-------------------------------------------------- ------------------
HNB-Blue purple 0.579 (650 nm)
+ Blue 1.119 (Same as above)
-------------------------------------------------- ------------------
TPC-Blue 0.573 (600nm)
+ Colorless 0.185 (same as above)
-------------------------------------------------- ------------------
MTB-light blue 1.153 (610 nm)
+ Dark blue 0.700 (same as above)
-------------------------------------------------- ------------------
XB-1-Orange 0.178 (600 nm)
+ Peach 0.444 (same as above)
-------------------------------------------------- ------------------
実施例２：ＮｉイオンとＣｈ−３添加による核酸増幅の検出
実施例１で用いたＬＡＭＰ用反応液（以下「ＬＡＭＰ用反応液」）に、ＮｉＣｌ2を０．１ｍＭおよびクロロホスフォナゾ−３（Ｃｈ−３）を４０μＭとなるようにそれぞれ添加し、実施例１に準じてＰＳＡのＤＮＡ増幅を行い、目視による増幅後の反応液の色調の観察とスペクトル測定を行った。
Example 2 Detection of Nucleic Acid Amplification by Addition of Ni Ion and Ch-3 In the reaction solution for LAMP used in Example 1 (hereinafter referred to as “LAMP reaction solution”), 0.1 mM of NiCl 2 and chlorophosphonazo-3 (Ch-3) was added to a concentration of 40 μM, DNA amplification of PSA was performed according to Example 1, and the color tone of the reaction solution after amplification was visually observed and the spectrum was measured.
その結果を図６に示す。ＮｉＣｌ2およびＣｈ−３が無添加のものは、ポジティブ、ネガティブにスペクトルの差はほとんど見られなかったが、ＮｉＣｌ2およびＣｈ−３を添加したものは、３００〜７００ｎｍ全域でスペクトルに大きな差が現れ、極大吸収波長５８０ｎｍでの吸光度は、ネガティブが１．３９０に対し、ポジティブが１．６７８、また、目視による増幅後の反応液の色調は、ネガティブが青紫色に対し、ポジティブが青色であり、増幅の有無を確認することができた。。 The result is shown in FIG. In the case where NiCl 2 and Ch-3 were not added, there was almost no difference in the spectrum between positive and negative, but in the case where NiCl 2 and Ch-3 were added, there was a large difference in the spectrum in the entire region of 300 to 700 nm. The absorbance at the maximum absorption wavelength of 580 nm is 1.390 for negative, 1.678 for positive, and the color of the reaction solution after visual amplification is blue for negative and blue for positive. The presence or absence of amplification could be confirmed. .
実施例３：ＭｎＣｌ 2 とカルセイン添加による核酸増幅の検出
ＬＡＭＰ用反応液に、ＭｎＣｌ2を１ｍＭおよびカルセインを２５μＭとなるようにそれぞれ添加し、実施例１に準じてＰＳＡのＤＮＡ増幅を行い、目視による増幅後の反応液の色調の観察とスペクトル測定を行った。
Example 3: MnCl 2 and detecting LAMP reaction solution for nucleic acid amplification by calcein addition, the MnCl 2 were added respectively 1mM and calcein as a 25 [mu] M, perform DNA amplification of PSA according to Example 1, visual The color tone of the reaction solution after amplification was measured and the spectrum was measured.
その結果を図７に示す。ＭｎＣｌ2およびカルセインが無添加のものは、ポジティブ、ネガティブにスペクトルの差はほとんど見られなかったが、ＭｎＣｌ2およびカルセインを添加したものは、極大吸収波長が、ネガティブが４９４ｎｍ（吸光度１．７１４）に対し、ポジティブでは４８９ｎｍ（吸光度１．６５９）とわずかに変化した。また、目視による増幅後の反応液の色調は、ネガティブがオレンジ色に対し、ポジティブが黄色となり、増幅の有無を確認することができた。ここで、増幅後の反応液について、ＵＶイルミネーターで波長３６５ｎｍの紫外線を照射すると、ポジティブのみで強い蛍光色が観察された。さらに、この反応液を蛍光光度計で測定すると、Ｅｘ．３６０ｎｍ（励起波長）−Ｅｍ．５１４ｎｍ（蛍光波長）で、蛍光強度がポジティブで２６３．８７、ネガティブで２４．２５となり、蛍光測定においても増幅の有無を確認することができた。図８（ネガティブ）および図９（ポジティブ）に蛍光スペクトル図を示す。 The result is shown in FIG. When MnCl 2 and calcein were not added, there was almost no difference in spectrum between positive and negative, but when MnCl 2 and calcein were added, the maximum absorption wavelength was 494 nm (absorbance 1.714). On the other hand, the positive value slightly changed to 489 nm (absorbance 1.659). In addition, the color tone of the reaction solution after amplification by visual observation was negative for orange and positive for yellow, confirming the presence or absence of amplification. Here, when the amplified reaction solution was irradiated with ultraviolet light having a wavelength of 365 nm with a UV illuminator, a strong fluorescent color was observed only in positive. Further, when this reaction solution was measured with a fluorometer, Ex. 360 nm (excitation wavelength)-Em. At 514 nm (fluorescence wavelength), the fluorescence intensity was 263.87 for positive and 24.25 for negative, and the presence or absence of amplification could be confirmed in the fluorescence measurement. FIG. 8 (negative) and FIG. 9 (positive) show fluorescence spectrum diagrams.
実施例４：核酸増幅反応の呈色を指標としたモニタリング
ＬＡＭＰ用反応液に、ＨＮＢまたはＢＴを１００μＭの濃度となるように添加し、測定波長（６５０ｎｍ）が固定式のリアルタイム濁度計ＬＡ−２００（ テラメックス社製 ) を用いて、ＰＳＡのＤＮＡ増幅反応を６５℃で５０分間行った。金属指示薬無添加のものをベースラインとし、増幅反応によって生じるＨＮＢまたはＢＴの色調変化に伴う吸光度変化をモニタリングした。
Example 4: To monitor LAMP reaction solution using coloration of nucleic acid amplification reaction as an index , HNB or BT was added to a concentration of 100 μM, and the measurement wavelength (650 nm) was a fixed real-time turbidimeter LA- 200 (manufactured by Teramex) was used for PSA DNA amplification reaction at 65 ° C. for 50 minutes. Changes in absorbance due to changes in the color tone of HNB or BT caused by the amplification reaction were monitored based on the baseline with no metal indicator added.
その結果を図１０に示す。ＨＮＢまたはＢＴのどちらを添加しても、１０分過ぎにポジティブのみ急激な吸光度の上昇がみられた。このことは、呈色反応のみによる増幅反応のモニタリングすなわちリアルタイム測定が可能であることを示唆する。 The result is shown in FIG. When either HNB or BT was added, only a positive increase in absorbance was observed after 10 minutes. This suggests that the amplification reaction can be monitored only by the color reaction, that is, real-time measurement is possible.
実施例５：核酸増幅反応の呈色と濁度を指標としたモニタリング
ＬＡＭＰ用反応液に、ＨＮＢまたはＢＴを１００μＭの濃度となるように添加し、実施例４で使用した濁度計を用いて、ＰＳＡのＤＮＡ増幅反応を６５℃で６０分間行い、増幅反応によって生じる色調と濁度の変化に伴う吸光度変化をモニタリングした。
Example 5: HNB or BT was added to the reaction solution for monitoring LAMP using the coloration and turbidity of the nucleic acid amplification reaction as indices, and the turbidimeter used in Example 4 was used. The PSA DNA amplification reaction was performed at 65 ° C. for 60 minutes, and the change in absorbance due to the change in color tone and turbidity caused by the amplification reaction was monitored.
その結果を図１１に示す。金属イオンとピロリン酸による濁度（図中「ポジティブ」）に、金属イオンと金属指示薬による呈色が加わり、より大きい吸光度の上昇（図中「ＢＴ−またはＨＮＢ−ポジティブ」）が見られた。このことは、より高感度なモニタリングが可能であると同時に、呈色または濁度のどちらの手段を用いても、核酸増幅の有無を検出することが可能であることを示唆している。 The result is shown in FIG. Addition of coloration due to metal ions and a metal indicator to turbidity due to metal ions and pyrophosphate (“positive” in the figure), and a greater increase in absorbance (“BT- or HNB-positive” in the figure) was observed. This suggests that more sensitive monitoring is possible, and at the same time, it is possible to detect the presence or absence of nucleic acid amplification using either coloration or turbidity.
実施例６：核酸増幅反応の蛍光によるモニタリング（１）
ＬＡＭＰ用反応液に、ＭｎＣｌ2を０．２５ｍＭおよびカルセインを５μＭ濃度となるように添加し、リアルタイム高温蛍光マイクロプレートリーダーＦｌｕｏＤｉａ Ｔ７０（ 大塚電子株式会社製 ）を用いて、ＰＳＡのＤＮＡ増幅反応を６５℃で６０分間行い、増幅反応によって生じる蛍光強度の変化をモニタリングした。
Example 6: Monitoring of nucleic acid amplification reaction by fluorescence (1)
To the reaction solution for LAMP, MnCl 2 was added to a concentration of 0.25 mM and calcein to a concentration of 5 μM, and a PSA DNA amplification reaction was performed using a real-time high-temperature fluorescent microplate reader FluoDia T70 (manufactured by Otsuka Electronics Co., Ltd.) The change was performed at 60 ° C. for 60 minutes, and the change in fluorescence intensity caused by the amplification reaction was monitored.
その結果を図１２に示す。Ｅｘ．４８５ｎｍ−Ｅｍ．５３０ｎｍにおいて、１３分過ぎにポジティブのみで急激な蛍光強度の上昇がみられた。このことより、金属イオンと金属指示薬の添加による蛍光測定によって、増幅反応のモニタリングが可能であることが示唆された。 The result is shown in FIG. Ex. 485 nm-Em. At 530 nm, a rapid increase in fluorescence intensity was observed only after 13 minutes. This suggests that the amplification reaction can be monitored by fluorescence measurement by adding metal ions and metal indicators.
実施例７：核酸増幅反応の蛍光によるモニタリング（２）
ＬＡＭＰ用反応液に、ＴＰＣを２００μＭおよびフルオレセインを６０ｎＭの濃度となるように添加し、ＡＢＩ ＰＲＩＳＭ ７７００ ＳＹＳＴＥＭ（ Perkin Elmer Applied Biosystems社製 ）を用いて、ＰＳＡのＤＮＡ増幅反応を６５℃で６０分間行い、増幅反応によって生じる蛍光強度の変化をモニタリングした。
Example 7: Monitoring of nucleic acid amplification reaction by fluorescence (2)
To the reaction solution for LAMP, 200 μM of TPC and fluorescein were added to a concentration of 60 nM, and PSA DNA amplification reaction was performed at 65 ° C. for 60 minutes using ABI PRISM 7700 SYSTEM (manufactured by Perkin Elmer Applied Biosystems). The change in fluorescence intensity caused by the amplification reaction was monitored.
その結果を図１３に示す。励起波長４８８ｎｍ、Ｄｙｅ Ｌａｙｅｒ ＦＡＭ (蛍光波長５２０〜５３０ｎｍ)において、１５分過ぎにポジティブのみで急激な蛍光強度の上昇がみられた。これは、核酸増幅の進行と共に、ＴＰＣによる反応液の色調が弱くなることで、反応前にＴＰＣに吸収されていたフルオレセインの蛍光色が次第に観察されるようになってくるためである。このことより、金属指示薬と蛍光物質の共存による蛍光測定によって、増幅反応のモニタリングが可能であることが示唆された。 The result is shown in FIG. At an excitation wavelength of 488 nm and Dye Layer FAM (fluorescence wavelength of 520 to 530 nm), a rapid increase in fluorescence intensity was observed only after 15 minutes. This is because the fluorescent color of fluorescein absorbed by TPC before the reaction is gradually observed as the nucleic acid amplification progresses and the color tone of the reaction solution by TPC becomes weaker. This suggests that the amplification reaction can be monitored by fluorescence measurement using a metal indicator and a fluorescent substance.
実施例８：核酸増幅反応の蛍光によるモニタリング（３）
ＬＡＭＰ用反応液に、ＭｎＣｌ2を０．５ｍＭおよびカルセインを２５μＭの濃度となるように添加し、サーマルサイクラー Ｔ ｇｒａｄｉｅｎｔ ( Biometra社製 )を用いて、ＰＳＡのＤＮＡ増幅反応を６５℃で３０分間行った。反応時間２分毎にデジタルカメラＱＶ−２９００ＵＸ ( CASIO社製 ）、ＵＶランプＵＶＧＬ−５８型ミネラライト・ランプ( UVP )を用い、図１５に示す装置構成で、反応液の蛍光をデジタルカメラ（ Shutter Speed 1/20sec. F 3.2 ）を固定したまま撮影を行った。
Example 8: Monitoring of nucleic acid amplification reaction by fluorescence (3)
To the reaction solution for LAMP, MnCl 2 was added to a concentration of 0.5 mM and calcein to a concentration of 25 μM, and a PSA DNA amplification reaction was performed at 65 ° C. for 30 minutes using a thermal cycler T gradient (manufactured by Biometra). It was. Using a digital camera QV-2900UX (manufactured by CASIO) and a UV lamp UVGL-58 type Mineralite lamp (UVP) every 2 minutes, the fluorescence of the reaction solution is measured with a digital camera (Shutter). I took a picture with
その結果を図１４に示す。図１４は、反応終了後、撮影した画像を画像編集ソフト（ Adobe Photo Shop 5.5 )で変換し、反応液部分のヒストグラムの平均値（チャンネル；ブラック）を算出し、得られた平均値を撮影時間に対してプロットしたグラフである。ポジティブのみ蛍光の上昇に伴うヒストグラム平均値の上昇が見られた。このことより、簡易装置を用いた増幅反応のリアルタイム測定が可能であることが示唆される。
The result is shown in FIG. FIG. 14 shows that after the reaction is completed, the photographed image is converted by image editing software (Adobe Photo Shop 5.5), the average value (channel; black) of the histogram of the reaction solution is calculated, and the obtained average value is taken as the photographing time. Is a graph plotted against. Only positives showed an increase in the average histogram value with an increase in fluorescence. This suggests that real-time measurement of the amplification reaction using a simple device is possible.
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