JP3969537B2 - Method for quantifying pyrophosphate and nucleic acid and reagent composition thereof - Google Patents

Description

【００５４】
以上のような方法で生成したホルマザンを定量することにより、ピロリン酸を定量することが可能となる。なお、ホルマザンの定量は公知の方法を用いて容易に行うことができる。
【００５５】
キサンチンデヒドロゲナーゼ（EC 1.1.1.204）およびキサンチンオキシダーゼ（EC 1.1.3.22）は同一の酵素であり、テトラゾリウム塩が存在する場合にはデヒドロゲナーゼとして作用し、水と酸素が存在するとオキシダーゼとして作用し、酸素が一旦スーパーオキサイドアニオン（O₂ ^-）になった後、速やかに水と反応して過酸化水素を発生する。
【００５６】
ピロリン酸定量操作として最も好ましい操作手順としては、ピロリン酸を含む試料に、キサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）およびイノシン酸（キサンチル酸が含まれていても、キサンチル酸のみでも良い）を同時に添加して、試料中に混在するヒポキサンチンおよび／またはキサンチンを除去し、次いでヒポキサンチングアニンホスホリボシルトランスフェラーゼ（EC 2.4.2.8）およびテトラゾリウム塩を同時に添加して、ピロリン酸をヒポキサンチンおよび／またはキサンチンに変換し、さらにホルマザンと尿酸にまで変換する方法である。この操作方法であれは、試薬類の添加工程は２回で済み効率的である。
【００５７】
本方法に用いるテトラゾリウム塩は特に制限されるものではなく、例えばパラヨードニトロテトラゾリウムバイオレット、チアゾイルブルー、ネオテトラゾリウムクロライド、ニトロブルーテトラゾリウム、テトラニトロブルーテトラゾリウム、テトラゾリウムブルー、テトラゾリウムレッド、テトラゾリウムバイオレット、チオカルバモイルニトロブルーテトラゾリウム、トリフェニルテトラゾリウムクロライド、ＷＳＴ−１、ＷＳＴ−３等を挙げることができる。
【００５８】
例えば、パラヨードニトロテトラゾリウムバイオレットを用いて、ピロリン酸を測定する場合には、通常は1〜100mM程度のイノシン酸、1〜100mM程度の塩化マグネシウム、1〜100 unit/L程度のキサンチンデヒドロゲナーゼ／オキシダーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）20μLをピロリン酸を含む試料100μLに添加し、27〜65℃で1〜10分程度反応させた後、0.1〜100mM程度のパラヨードニトロテトラゾリウムバイオレット、1〜100mM程度の塩化マグネシウム、10〜1000 unit/mL程度のヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）80μL を加え、27〜65度で10〜120分程度反応させた後、400-550nm程度の波長で比色定量すれば良い。
【００５９】
次に本発明により提供される第２のピロリン酸測定方法について説明する。 第２の方法は、上記第１の方法と同様、ピロリン酸を含む測定試料に必要量のキサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）を添加して、試料中に予め混在していたヒポキサンチンおよび／またはキサンチンを除去する工程を具備する。次いで必要量のイノシン酸（キサンチル酸が含まれていても、キサンチル酸のみでも良い）およびヒポキサンチングアニンホスホリボシルトランスフェラーゼ（EC 2.4.2.8）を添加して、試料中のピロリン酸をヒポキサンチンおよび／またはキサンチンに変化させる。次いで、キサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）およびスーパーオキサイドアニオン（O₂ ^-）と反応して化学発光性を示す化学発光試薬を添加して、ピロリン酸由来のヒポキサンチンおよび／またはキサンチンを光と尿酸に変換する。この一連の反応は以下のように示される。
【００６０】
【化２】

【００６１】
ヒポキサンチンにキサンチンオキシダーゼが作用すると、酸素が一旦スーパーオキサイドアニオン（O₂ ^-）になった後、速やかに水と反応して過酸化水素を発生する。しかし、化学発光試薬が存在する場合は、スーパーオキサイドアニオン（O₂ ^-）は化学発光試薬と反応し、発光を示す。化学発光試薬としては、スーパーオキサイドアニオン（O₂ ^-）に対して特異的に発光を示す化合物が好ましく、特に過酸化水素と反応しないことが重要である。この目的のために好適な化学発光試薬としては、特開平５−６０６９７号公報に開示されたウミホタルルシフェリン誘導体や特開平１１−２０９３７９号公報に開示されたイミダゾヒラジノン誘導体などを挙げることができる。
【００６２】
ピロリン酸定量操作として最も好ましい操作手順としては、ピロリン酸を含む試料に、キサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）およびイノシン酸（キサンチル酸が含まれていても、キサンチル酸のみでも良い）を同時に添加して、試料中に混在するヒポキサンチンおよび／またはキサンチンを除去し、次いでヒポキサンチングアニンホスホリボシルトランスフェラーゼ（EC 2.4.2.8）および化学発光試薬を同時に添加して、ピロリン酸をヒポキサンチンおよび／またはキサンチンに変換し、さらに光と尿酸にまで変換する方法である。この操作方法であれは、試薬類の添加工程は２回で済み効率的である。
【００６３】
例えば、ウミホタルルシフェリン誘導体の一種である２−メチル−６−（４−メトキシフェニル）−３，７−ジヒドロイミダゾ[１，２−a]ピラジン−３−オン ハイドロクロライド（MCLA）を用いて、ピロリン酸を測定する場合には、通常は1〜100mM程度のイノシン酸、1〜100mM程度の塩化マグネシウム、1〜100 unit/L程度のキサンチンデヒドロゲナーゼ／オキシダーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）20μLをピロリン酸を含む試料100μLに添加し、27〜65℃で1〜10分程度反応させた後、0.1〜100μM程度のMCLA、1〜100mM程度の塩化マグネシウム、10〜1000 unit/mL程度のヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）80μL を加え、27〜65度で10〜120分程度反応させた後、反応液の化学発光強度測定すれば良い。
【００６４】
この方法はホルマザンでの呈色反応を利用したピロリン酸測定方法の検出限界が高いことから、検出限界の低い場合に化学発光検出を用いることによりピロリン酸の検出感度を上昇させることを目的として案出した。
【００６５】
次に本発明に係る第３の方法について説明する。第３の方法は、前記第１及び第２の方法と同様、ピロリン酸を含む測定試料に必要量のキサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）を添加して、試料中に予め混在していたヒポキサンチンおよび／またはキサンチンを除去する。次いで必要量のイノシン酸（キサンチル酸が含まれていても、キサンチル酸のみでも良い）およびヒポキサンチングアニンホスホリボシルトランスフェラーゼ（EC 2.4.2.8）を添加して、試料中のピロリン酸をヒポキサンチンおよび／またはキサンチンに変化させる。次いで、キサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）およびスーパーオキサイドアニオン（O₂ ^-）と反応して蛍光を示す蛍光試薬を添加して、ピロリン酸由来のヒポキサンチンおよび／またはキサンチンを蛍光物質と尿酸に変換する。この一連の反応は以下のように示される。
【００６６】
【化３】

【００６７】
ヒポキサンチンにキサンチンオキシダーゼが作用すると、酸素が一旦スーパーオキサイドアニオン（O₂ ^-）になった後、速やかに水と反応して過酸化水素を発生する。しかし、スーパーオキサイドアニオン（O₂ ^-）検出用の蛍光試薬が存在する場合は、スーパーオキサイドアニオン（O₂ ^-）は蛍光試薬と反応し、蛍光を示す。蛍光試薬としては、スーパーオキサイドアニオン（O₂ ^-）に対して特異的に発光を示す化合物が好ましく、特に過酸化水素と反応しないことが重要である。この目的のために好適な蛍光試薬としては、Amplex^TM Red（Anal. Biochem., 253, 162-168 (1997)もしくはJ. Immunol. Methods 202, 133-141 (1997)）に開示された蛍光試薬などを挙げることができる。
【００６８】
ピロリン酸定量操作として最も好ましい操作手順としては、ピロリン酸を含む試料に、キサンチンデヒドロゲナーゼ（EC 1.1.1.204）／キサンチンオキシダーゼ（EC 1.1.3.22）およびイノシン酸（キサンチル酸が含まれていても、キサンチル酸のみでも良い）を同時に添加して、試料中に混在するヒポキサンチンおよび／またはキサンチンを除去し、次いでヒポキサンチングアニンホスホリボシルトランスフェラーゼ（EC 2.4.2.8）および蛍光試薬を同時に添加して、ピロリン酸をヒポキサンチンおよび／またはキサンチンに変換し、さらに蛍光化合物と尿酸にまで変換する方法である。この操作方法であれは、試薬類の添加工程は２回で済み効率的である。
【００６９】
例えば、Amplex^TM Redを用いて、ピロリン酸を測定する場合には、通常は1〜100mM程度のイノシン酸、1〜100mM程度の塩化マグネシウム、1〜100 unit/L程度のキサンチンデヒドロゲナーゼ／オキシダーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）20μLをピロリン酸を含む試料100μLに添加し、27〜65℃で1〜10分程度反応させた後、0.1〜1000μM程度のAmplex^TM Red、1〜100mM程度の塩化マグネシウム、10〜1000 unit/mL程度のヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む5〜500mM程度のトリス塩酸緩衝液（pH 6.5〜8.5程度）80μL を加え、27〜65度で10〜120分程度反応させた後、反応液の蛍光強度（励起波長550〜570nm、蛍光波長580〜610nm）を測定すれば良い。
【００７０】
この方法はホルマザンでの呈色反応を利用したピロリン酸測定方法の検出限界が高いことから、検出限界の低いに蛍光検出を用いることによりピロリン酸の検出感度を上昇させることを目的として案出した。
【００７１】
本発明のピロリン酸測定方法は、ばらばらの供給源から得られる必要な試薬及び材料を用いて実施することは可能であるが、本発明の測定方法に有用な試薬及び酵素等が試薬キットの一部として供給されることが有利である。本発明での測定に使用される酵素類、コファクター類、緩衝液類、塩類の最低量、および各々の適当な範囲は当該技術分野で周知であり、本発明により提供される試薬キットに含まれる構成品は、所定の方法に適するいずれかの濃度で提供および使用され、予め蓋付き容器に分注されていることが望ましい。本発明の試薬キットに含まれる構成品は、測定試料及び測定方法等に応じて必要とされる試薬及び酵素各々を単独で含有するもの、あるいは２種以上の試薬又は酵素等の混合物であり得る。また、本発明の試薬キットに含まれる構成品は、試料に対して別個に添加または混合してから添加することができる。これらの材料は、必要な安定性、安全性及び取り扱いの簡便さの目的で適宜包装（乾式又は湿式）することができる。もしくは、必要な試薬類が固定された濾紙などの試薬支持体を構成品として含む試薬キットも本発明の測定方法において有意に用いることができ、該試薬支持体上で上記反応を行わせることも可能である。本試薬キットを用いて、ピロリン酸を検知する手段としては、紫外可視分光光度計、蛍光分光光度計、化学発光検出器などの高度な光学機器を用いた精密定量の他に、ピロリン酸の有無を確認するための簡易判定として、肉眼による色見本との濃淡比較もしくは写真やCCDカメラ等による呈色または蛍光、発光の濃淡によっておおよその濃度を知ることも可能である。
【００７２】
また、本発明のピロリン酸測定方法を用いて、核酸の有無の検出または定量を行おうとする場合に用いられる試薬及び酵素は、試薬キットの構成品として供給されることが有利である。このような試薬キットの構成品としては、上記ピロリン酸測定用の試薬キットに含まれる構成品の他に、更に核酸を増幅するために必要な試薬を含む。核酸を増幅するために必要な試薬としては、プライマー類、コファクター類、ヌクレオチド−３−リン酸類等を挙げることができる。核酸増幅に使用されるプライマー類、コファクター類、ヌクレオチド−３−リン酸類の最低量、および各々の適当な範囲は当該技術分野で周知であり、これらは所定の方法に適するいずれかの濃度で提供および使用される。
【００７３】
核酸増幅法として、PCR反応の他に、LAMP法やICAN法など幾つかの手法があるが、核酸増幅に伴い、ピロリン酸が同時に生成される場合は、本発明の手法は、PCR以外の方法にも同様に適用できる。色素生成による判定では、特別に検出装置を使わず、既存の核酸増幅試薬に本発明のピロリン酸測定方法に有用な上記試薬を添加すれば、特定の核酸の存在の有無が目視で判定できる。
【００７４】
エンドポイント分析によって、核酸増幅反応の有無を検知したい場合は、試料中に予め含有されているヒポキサンチンおよび／またはキサンチンを除去する工程（最も好ましい測定方法として上述した操作手順においては２工程にわたる試薬類の添加工程のうち１回目の添加工程）は、上述のいずれかの核酸増幅反応の開始前、開始後のどちらでもよい。その場合核酸増幅反応において９５℃以上の温度を加えても酵素活性を失わない耐熱性のキサンチンデヒドロゲナーゼ／オキシダーゼを用いることで、ピロリン酸検出のための前記除去工程後の工程（２工程にわたる添加工程にあっては２回目の添加工程）において再度キサンチンデヒドロゲナーゼ／オキシダーゼを添加する必要はない。リアルタイム分析によって、核酸増幅反応の有無を検知したい場合は、ピロリン酸検出試薬のうち、キサンチンデヒドロゲナーゼ／オキシダーゼおよびヒポキサンチングアニンホスホリボシルトランスフェラーゼの両方の酵素が耐熱性であることが必要である。
【００７５】
ＰＣＲ反応は以下の様に反応しピロリン酸を副生成物として遊離する。従って、ピロリン酸（ＰＰｉ）生成量は増幅DNA量に比例する。
【００７６】
【化４】

【００７７】
本発明によりウイルス等の初期鋳型核酸量を定量する場合は、ＰＣＲによって指数関数的にピロリン酸が生成することを利用する。ＰＣＲ反応生成物は、反応初期にはほぼ指数関数的増加を示し、十分なサイクル数ＰＣＲを行った場合は、初期鋳型核酸量に関係なくほぼ同じ量のＰＣＲ反応生成物が得られる。従ってＰＣＲ反応生成物がある一定量に達するサイクル数（ＣＴ）は初期鋳型核酸量と逆相関の関係にある。またこの関係が成り立つためには初期鋳型核酸量が異なっていても指数増幅期の増幅率が等しくなければならない。標的配列が同じ場合はこの関係が成立しているため、ＣＴから初期鋳型核酸量を推定することが可能である。本発明ではサイクル数を変化させてＰＣＲ反応を行い、各サイクル数におけるピロリン酸の生成量から初期鋳型核酸量を定量する方法を用いる。例えばパソコンによる色データの時間変化解析などにより、おおよそのピロリン濃度とその時間変化から、初期鋳型核酸量を定量することができる。
【００７８】
【実施例】
次に本発明を実施例により更に詳細に説明するが、実施例は例示のために提示するものであって、本発明を限定することを意図するものではない。
【００７９】
（実施例１）
初めに次の２種類の試薬を予め調製しておく。
【００８０】
（１）20mMイノシン酸、10mM塩化マグネシウム、0.4units/mLキサンチンデヒドロゲナーゼ／オキシダーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
（２）4mMパラヨードニトロテトラゾリウムバイオレット、10mM塩化マグネシウム、100units/mLヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
既知濃度のピロリン酸溶液100μlに（１）を20μl加え、６０℃で１分間反応させた後、（２）を80μl加え、６０℃で１０分間反応させた。この反応液90μlを９６穴のマイクロプレートに取り、マイクロプレートリーダー「Bio-Tek Elx 800」（Bio-Tek Instruments製）を用いて、515nmと630nmの吸光度を測定した。そのときのピロリン酸の検量線を図１に示す。尚、図１における吸光度は515nmにおける吸光度から630nmにおける吸光度を引くことによって求めた。本実施例の結果から、本発明の方法を用いて精度良くピロリン酸を測定できることがわかる。
【００８１】
（実施例２）
実施例１と同様の試薬を調製し、10%および50%の牛血清または1〜10 mMのリン酸カリウム(KH₂PO₄)、1〜10 mMのATP、50〜500 mMのヒポキサンチン、50〜500 mMのキサンチンを含む500μMのピロリン酸を含む１０ｍＭ Tris HCl溶液（ｐＨ７．８）100μlに（１）を20μl加え、６０℃で１分間反応させた後、（２）を80μl加え、６０℃で１０分間反応させた。この反応液90μlを９６穴のマイクロプレートに取り、マイクロプレートリーダー「Bio-Tek Elx 800」（Bio-Tek Instruments製）を用いて、515nmと630nmの吸光度を測定した。そのときの吸光度を表１に示す。尚、表１における吸光度は515nmにおける吸光度から630nmにおける吸光度を引くことによって求めた。本実施例の結果から、本発明の方法を用いた場合、生体サンプルおよび各種生体サンプル中に存在するリン酸やATPに影響されることなく、ピロリン酸を測定できることがわかる。また、バックグラウンド原因物質のヒポキサンチンとキサンチンは、試料中に50〜100 mM程度存在しても測定値に影響を与えなかった。もし100 mM以上のヒポキサンチンやキサンチンが存在する試料を測定する場合には、（１）液中のキサンチンデヒドロゲナーゼ／オキシダーゼの濃度を多くしたり、（１）液を加えた後の反応時間を長くすればよいことは明らかである。
【００８２】
【表１】

【００８３】
（実施例３）
アマシャムファルマシアバイオテク（株）のReady-To-Go PCR Breadsを用いて、PCR法による核酸の増幅を行った。PCRを行う反応溶液はキット添付のControl Lambda DNA および１組のプライマーを用いて、キット添付のプロトコールに従って調製した。そして、変性（95℃、１分）、アニーリング（55℃、１分）、DNA伸長反応（72℃、１分）のサイクルを、０、１０、２０、２５、２７、３０、３５サイクル行った。反応溶液の組成は以下のとおりである。
【００８４】
反応溶液の組成（PCR Breads １ビーズ中）
Control Lambda DNA １μｌ(0または1ng/μlのもの)
Control Primer 1 １μｌ
Control Primer 2 １μｌ
滅菌蒸留水 ２２μｌ
PCR終了後、50μlの反応溶液に対して、実施例１と同様の方法を用いて比色定量した。そのときの吸光度とサイクル数の関係を図2に示す。この図から、サイクル数の増加に伴って吸光度が上昇するため、本測定方法を用いて目的核酸の有無の判断が可能となることがわかる。
【００８５】
（実施例４）
初めに次の２種類の試薬を予め調製しておく。
【００８６】
（１）20mMイノシン酸、10mM塩化マグネシウム、0.4units/mLキサンチンデヒドロゲナーゼ／オキシダーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
（２）32μMのMCLA、10mM塩化マグネシウム、100units/mLヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
既知濃度のピロリン酸溶液50μlをマイクロチューブに取り、そこに（１）を10μl加え、６０℃で１分間反応させた後、（２）を40μl加え、６０℃で反応させながら１０分間、化学発光強度（化学発光検出器・浜松ホトニクス）を測定した。そのときのピロリン酸の検量線を図３に示す。本実施例の結果から、本発明の方法を用いて精度良くピロリン酸を測定できることがわかる。
【００８７】
（実施例５）
アマシャムファルマシアバイオテク（株）のReady-To-Go PCR Breadsを用いて、PCR法による核酸の増幅を行った。PCRを行う反応溶液はキット添付のControl Lambda DNA（0、0.1、1、10 ng/μlに調製して使用した。） および１組のプライマーを用いて、キット添付のプロトコールに従って調製した。そして、変性（95℃、１分）、アニーリング（55℃、１分）、DNA伸長反応（72℃、１分）のサイクルを、０、１０、１５、２０、２５、２７サイクル行った。反応溶液の組成は以下のとおりである。
【００８８】
反応溶液の組成（PCR Breads １ビーズ中）
Control Lambda DNA １μｌ（４種類の濃度に調製）
Control Primer 1 １μｌ
Control Primer 2 １μｌ
滅菌蒸留水 ２２μｌ
PCR終了後、25μlの反応溶液に対して、実施例４と同様の方法を用いて化学発光強度を測定した。そのときの化学発光強度とサイクル数の関係を図４に示す。この図から、サイクル数の増加に伴って化学発光強度が上昇するため、本測定方法を用いて目的核酸の有無の判断および初期鋳型核酸量の推定が可能となることがわかる。
【００８９】
（実施例６）
初めに次の２種類の試薬を予め調製しておく。
【００９０】
（１）20ｍＭイノシン酸、10mM塩化マグネシウム、0.4units/mLキサンチンデヒドロゲナーゼ／オキシダーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
（２）200μＭのAmplex^TM Red、10mM塩化マグネシウム、100units/mLヒポキサンチングアニンホスホリボシルトランスフェラーゼを含む100mMトリス塩酸緩衝液（pH 7.6）
既知濃度のピロリン酸溶液50μlをマイクロチューブに取り、そこに（１）を10μl加え、６０℃で１分間反応させた後、（２）を40μl加え、６０℃で１０分間反応させた後、蛍光強度（蛍光分光光度計・日本分光）を測定した。そのときのピロリン酸の検量線を図５に示す。本実施例の結果から、本発明の方法を用いて精度良くピロリン酸を測定できることがわかる。
【００９１】
（実施例７）
培養したSalmonella Tonpson, Salmonella Enteritidis, 大腸菌Ｏ6及びＯ148を熱水抽出後、サルモネラinvA遺伝子検出用プライマー（タカラバイオ）を用いてPCR反応を行い、反応終了後、実施例３と同様な方法でピロリン酸検出を行った結果、S. Tonpson, S. Enteritidisのみ呈色した（表２）。アガロースゲル電気泳動でPCR反応物の生成が確認された反応液中のピロリン酸量は、200-400μM程度であった。このように本法を利用することで、毒性のない試薬を用いかつ目視簡易判定により、細菌の同定を行うことができた。
【００９２】
【表２】

【００９３】
【発明の効果】
以上詳述したように、本発明は従来のピロリン酸定量法の欠点であるバックグラウンドの上昇などを克服した簡便かつ精度の高いピロリン酸定量方法及び該定量方法に有用な試薬キットであり、本発明により特定核酸の有無の判別が正確かつ簡便に行うことが可能となった。
【図面の簡単な説明】
【図１】 本発明のピロリン酸定量方法を用いて得られたピロリン酸濃度と吸光度との関係を示す図。
【図２】 本発明のピロリン酸定量方法を用いて得られたサイクル数と吸光度との関係を示す図。
【図３】 本発明のピロリン酸定量方法を用いて得られたピロリン酸濃度と化学発光強度の関係を示す図。
【図４】 本発明のピロリン酸定量方法を用いて得られたサイクル数と化学発光強度の関係を示す図。
【図５】 本発明のピロリン酸定量方法を用いて得られたピロリン酸濃度と蛍光強度の関係を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting and quantifying pyrophosphate. The present invention also relates to a nucleic acid detection / quantification method using the pyrophosphate detection / quantification method, and more specifically, generated when a specific nucleic acid region is amplified using various nucleic acid amplification methods including a polymerase chain reaction. The present invention relates to a method for detecting and quantifying nucleic acids by detecting and quantifying pyrophosphate.
[0002]
[Prior art]
Pyrophosphate is a substance that is released by various enzyme reactions existing in the living body. Therefore, various measuring methods for pyrophosphate have been developed. Among them, as a simpler method, pyrophosphoric acid is converted to ATP with adenosine triphosphate (ATP) sulfurylase, and the generated ATP is measured (for example, see Non-Patent Document 1), or pyrophosphate is converted to inorganic pyrophosphatase. In general, a method of decomposing into phosphoric acid and measuring the generated phosphoric acid (see, for example, Non-Patent Document 2) is widely used. In addition to these, various techniques have been reported regarding the method for measuring pyrophosphate (see, for example, Non-Patent Documents 3-5).
[0003]
Nucleic acid polymerase, which is one of the enzymatic reactions that liberate pyrophosphate, is used in nucleic acid amplification techniques that detect a small amount of nucleic acid by continuously amplifying a specific base sequence. For example, the polymerase chain reaction method (PCR method) amplifies a specific nucleic acid region in a nucleic acid molecule several million times in a test tube, and various techniques have been disclosed for the PCR method (for example, , See Patent Documents 1-3). By using this PCR method, it is possible to detect a specific nucleic acid region of the target nucleic acid in an amplified state.
[0004]
As a general method for detecting the amplified specific nucleic acid region, the reaction solution after completion of PCR is separated by agarose electrophoresis and then stained and discriminated by the size of the band (molecular weight). A method of detecting a solution by a dot hybridization method has been performed. This detection method is characterized by being able to directly amplify and detect DNA in a sample, so that the test time is short and gene identification is easy. However, the complicated and time-consuming electrophoresis process is often inconvenient for on-site use.
[0005]
As a method for detecting and quantifying PCR products without performing electrophoresis, a reagent that emits fluorescence only when intercalated with DNA is added to the reaction solution before starting PCR, and a fluorescence spectrophotometer is used. For example, there is a method for measuring the amount of amplified DNA by measuring the fluorescence intensity (see, for example, Patent Document 4). The initial template amount of the target nucleic acid can be obtained from the change by performing PCR in the presence of an intercalating fluorescent dye and measuring the fluorescence of the reaction solution for each PCR cycle. However, an apparatus for measuring the fluorescence intensity designed for such a purpose every cycle has the disadvantage of being expensive. Also, reagents that intercalate with DNA are harmful to living organisms.
[0006]
As a method not using an intercalating reagent with DNA, for example, pyrophosphate as a by-product generated in the nucleic acid amplification process by PCR is led to ATP by the action of ATP sulfurylase, and this ATP is further converted by luciferin-luciferase method. A method for detecting pyrophosphate by emitting light and detecting it has been reported (see, for example, Patent Document 5 and Non-Patent Documents 6 and 7).
[0007]
In addition, a method for detecting a target gene nucleic acid by detecting phosphate, which is a product obtained by decomposing pyrophosphate using inorganic pyrophosphatase has been reported (for example, Patent Document 6, Non-Patent Document 8, 9).
[0008]
Furthermore, pyrophosphoric acid is converted into hypoxanthine using hypoxanthine guanine phosphoribosyltransferase, and the hypoxanthine is converted into formazan, hydrogen peroxide or superoxide anion using xanthine dehydrogenase / oxidase to detect these. (For example, refer to Patent Document 7 and Non-Patent Document 10).
[0009]
[Patent Document 1]
US Pat. No. 4,683,195
[0010]
[Patent Document 2]
US Pat. No. 4,683,202
[0011]
[Patent Document 3]
US Pat. No. 4,965,188
[0012]
[Patent Document 4]
JP-A-5-237000
[0013]
[Patent Document 5]
International Publication No. 92/16654 Pamphlet
[0014]
[Patent Document 6]
JP 7-59600 A
[0015]
[Patent Document 7]
Japanese Patent Application No. 2001-377229
[0016]
[Non-Patent Document 1]
"Analycal Biochemistry", 1985, vol. 151, p. 504-509
[0017]
[Non-Patent Document 2]
“Analycal Biochemistry”, 1997, Vol. 254, p. 18-22
[0018]
[Non-Patent Document 3]
“Analycal Biochemistry”, 1979, Vol. 94, p. 117-120
[0019]
[Non-Patent Document 4]
“Biochemical Journal”, 1985, Vol. 230, p. 255-260
[0020]
[Non-Patent Document 5]
“Analycal Biochemistry”, 1996, Vol. 243, p. 41-45
[0021]
[Non-Patent Document 6]
“Journal of Immunological Methods”, 1992, 156, p. 55-60
[0022]
[Non-Patent Document 7]
“Analycal Biochemistry”, 2001, 288, p. 28-38
[0023]
[Non-Patent Document 8]
“Analycal Biochemistry”, 1997, Vol. 254, p. 18-22
[0024]
[Non-patent document 9]
“Bio Techniques”, 1998, Vol. 25, p. 608-614
[0025]
[Non-Patent Document 10]
“The 49th Annual Meeting of the Japan Society for Food Science and Technology”, August 29, 2002, p. 119
[0026]
[Problems to be solved by the invention]
All of the above-described conventional methods for measuring pyrophosphate are detected by inducing pyrophosphate to ATP, phosphoric acid or hypoxanthine, so there is a high possibility that those substances present in the living body will be detected as the background. . Phosphoric acid is often used in foods, culture media, enzyme extraction buffers, and the like, and ATP is always present in living organisms such as bacteria and is a substance that is difficult to remove. Furthermore, ATP is a substrate for many enzyme reactions such as acetyl coenzyme A synthetase, and thus cannot be used for measuring the activity of certain enzyme reactions. Moreover, hypoxanthine may exist in fish and shellfish with poor freshness.
[0027]
The present invention was devised in view of the above problems, and provides a highly accurate method for detecting and quantifying pyrophosphate, and various nucleic acid amplification methods using the method for detecting and quantifying pyrophosphate. It is an object of the present invention to provide a method for detecting and quantifying nucleic acid by the method and a reagent kit useful for the method.
[0028]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have changed the order of addition of enzymes and reagents in a pyrophosphate detection / quantification method using hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase. Thus, it was newly found that the amount of pyrophosphate can be measured without being affected by the background substance. By detecting and quantifying pyrophosphate using this method, it is possible to detect and quantify pyrophosphate stably and with high accuracy. Furthermore, this pyrophosphate detection and quantification method can be used for nucleic acid detection and quantification by various nucleic acid amplification methods. The present invention was found to be extremely useful, and the present invention has been completed.
[0029]
That is, the present invention has the following configuration.
[0031]
  (1)  A pyrolin comprising the steps of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and adding a tetrazolium salt and hypoxanthine guanine phosphoribosyltransferase Acid detection and quantification method.
[0033]
  (2)  A step of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and a step of adding a chemiluminescent reagent for detecting a superoxide anion and hypoxanthine guanine phosphoribosyltransferase A method for detecting and quantifying pyrophosphate.
[0035]
  (3)  Adding a step of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and a step of adding a fluorescent reagent for detecting a superoxide anion and hypoxanthating anine phosphoribosyltransferase. A method for detecting and quantifying pyrophosphate.
[0036]
  (4)  A method for detecting and quantifying nucleic acid, the step of amplifying nucleic acid in a sample solution, and pyrophosphate contained in the sample solution after amplification (1) to(3)A method for detecting and quantifying a nucleic acid, comprising the step of detecting and quantifying using the method according to any one of the above.
[0037]
  (5)  Hypoxanthine guanine phosphoribosyl that does not lose its enzyme activity even when a temperature of 95 ° C or higher is appliedTransferaseAnd / or using xanthine dehydrogenase / oxidase, (1) to(3)The method for detecting and quantifying pyrophosphate according to any one of the above.
[0038]
  (6)  Using hypoxanthine guanine phosphoribosyl and / or xanthine dehydrogenase / oxidase, which does not lose its enzyme activity when a temperature of 95 ° C. or higher is applied,(4)The method for detecting and quantifying nucleic acids described in 1.
[0039]
  (7)  (1)A reagent kit useful for the detection and quantification method of pyrophosphate described in 1., wherein pyrophosphate and inosinic acid and / or xanthylic acid, tetrazolium salt, hypoxanthine guanine phosphoribosyltransferase, and xanthine dehydrogenase / oxidase are each independently A reagent kit containing as a reagent to be contained or as a mixed reagent of any two or more.
[0040]
  (8)  (2)A reagent kit useful for the method for detecting and quantifying pyrophosphate according to claim 1, comprising a chemiluminescent reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, hypoxanthine anine phosphoribosyltransferase and xanthine dehydrogenase / A reagent kit containing oxidase as a reagent containing each singly or as a mixed reagent of any two or more.
[0041]
  (9)  (3)A reagent kit useful for the method for detecting and quantifying pyrophosphate described in 1., a fluorescent reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, hypoxanthine guanine phosphoribosyltransferase, and xanthine dehydrogenase / oxidase And a reagent kit containing each of them alone or as a mixed reagent of any two or more thereof.
[0042]
  (10)  A reagent kit useful for detection and quantification of target nucleic acids,(7)Thru(9)A reagent kit comprising a reagent necessary for amplifying a nucleic acid in addition to the reagent kit according to any one of the above.
[0043]
  (11)  (1)A reagent kit useful for the pyrophosphate detection / quantification method according to claim 1, wherein pyrophosphate and inosinic acid and / or xanthylic acid, tetrazolium salt, hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase are immobilized A reagent kit comprising a support.
[0044]
  (12)  (2)A reagent kit useful for the method for detecting and quantifying pyrophosphate according to claim 1, comprising a chemiluminescent reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, hypoxanthine anine phosphoribosyltransferase and xanthine dehydrogenase / A reagent kit comprising a reagent support on which oxidase is immobilized.
[0045]
  (13)  (3)A reagent kit useful for the method for detecting and quantifying pyrophosphate described in 1., a fluorescent reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase A reagent kit comprising a reagent support on which is fixed.
[0046]
  (14)  Reagents necessary for further amplifying nucleic acids are fixed to the reagent support.(11)~(13)The reagent kit according to any one of the above.
[0047]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0048]
The measurement object of the present invention is pyrophosphate. The method of the present invention was developed by the present inventors as part of developing methods for measuring pyrophosphate produced by various nucleic acid amplification techniques. Therefore, changes in the amount of nucleic acid amplified using various methods such as PCR, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN), and loop-mediated isothermal amplification (LAMP) It is suitable as a method for measuring pyrophosphate when estimating.
[0049]
The pyrophosphate measuring method of the present invention is also effective as a measuring method for various pyrophosphates added to foods as food additives.
[0050]
As described above, pyrophosphate has conventionally been measured by, for example, a method using hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase. However, since the intermediate product hypoxanthine in this measurement system is present in meat and fish, there is a drawback that the background increases when this method is applied to the detection of nucleic acids in foods.
[0051]
The present inventors have newly found that it is possible to measure the amount of pyrophosphate without being affected by the background substance by changing the order of addition of the enzymes and reagents, which will be described in detail below. The present invention has been completed.
[0052]
In the first method for measuring pyrophosphate newly provided by the present invention, a necessary amount of xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) is added to a measurement sample containing pyrophosphate. And a step of removing hypoxanthine and / or xanthine previously mixed in the sample. Next, the necessary amount of inosinic acid (which may contain xanthylic acid or only xanthylic acid) and hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) are added, and pyrophosphate in the sample is converted into hypoxanthine and Change to xanthine. Xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and tetrazolium salt are then added to convert hypoxanthine and / or xanthine from pyrophosphate into formazan. This series of reactions is shown as follows.
[0053]
[Chemical 1]

[0054]
By quantifying formazan produced by the method as described above, pyrophosphate can be quantified. Formazan can be easily determined using a known method.
[0055]
Xanthine dehydrogenase (EC 1.1.1.204) and xanthine oxidase (EC 1.1.3.22) are the same enzymes, acting as dehydrogenase in the presence of tetrazolium salt, acting as oxidase in the presence of water and oxygen, Once superoxide anion (O₂ ^-) Immediately reacts with water to generate hydrogen peroxide.
[0056]
The most preferred procedure for pyrophosphate determination is that the sample containing pyrophosphate contains xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and inosinic acid (xanthylic acid, Acid may be added at the same time to remove hypoxanthine and / or xanthine present in the sample, then hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) and tetrazolium salt are added at the same time to add pyrophosphate Is converted to hypoxanthine and / or xanthine, and further converted to formazan and uric acid. Even if this operation method is used, the steps of adding the reagents need only be performed twice, which is efficient.
[0057]
The tetrazolium salt used in this method is not particularly limited. For example, paraiodonitrotetrazolium violet, thiazoyl blue, neotetrazolium chloride, nitroblue tetrazolium, tetranitroblue tetrazolium, tetrazolium blue, tetrazolium red, tetrazolium violet, thiocarbamoyl Examples thereof include nitro blue tetrazolium, triphenyltetrazolium chloride, WST-1, and WST-3.
[0058]
For example, when measuring pyrophosphate using paraiodonitrotetrazolium violet, usually about 1 to 100 mM inosinic acid, about 1 to 100 mM magnesium chloride, about 1 to 100 unit / L xanthine dehydrogenase / oxidase After adding 20 μL of about 5 to 500 mM Tris-HCl buffer (pH 6.5 to 8.5) containing 100 μL to a sample containing pyrophosphate and reacting at 27 to 65 ° C. for about 1 to 10 minutes, about 0.1 to 100 mM Add 80 μL of para-iodonitrotetrazolium violet, about 1 to 100 mM magnesium chloride, about 5 to 500 mM Tris-HCl buffer solution (about pH 6.5 to 8.5) containing about 10 to 1000 unit / mL hypoxanthine guanine phosphoribosyltransferase, After reacting at 27 to 65 degrees for about 10 to 120 minutes, colorimetric determination may be performed at a wavelength of about 400 to 550 nm.
[0059]
Next, the second pyrophosphoric acid measurement method provided by the present invention will be described. The second method is similar to the first method described above, in which a necessary amount of xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) is added to the measurement sample containing pyrophosphate, A step of removing the mixed hypoxanthine and / or xanthine. The required amount of inosinic acid (which may contain xanthylic acid or xanthylic acid alone) and hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) is then added to reduce the pyrophosphate in the sample to hypoxanthine and / or Or change to xanthine. Xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and superoxide anion (O₂ ^-And a chemiluminescent reagent which shows chemiluminescence is added to convert pyroxanthine-derived hypoxanthine and / or xanthine into light and uric acid. This series of reactions is shown as follows.
[0060]
[Chemical 2]

[0061]
When xanthine oxidase acts on hypoxanthine, oxygen once becomes superoxide anion (O₂ ^-) Immediately reacts with water to generate hydrogen peroxide. However, if a chemiluminescent reagent is present, the superoxide anion (O₂ ^-) Reacts with a chemiluminescent reagent and emits light. Chemiluminescent reagents include superoxide anions (O₂ ^-) Is preferable, and it is important that the compound does not react with hydrogen peroxide. Suitable chemiluminescent reagents for this purpose include, for example, Cypridina luciferin derivatives disclosed in JP-A-5-60697 and imidazohiradinone derivatives disclosed in JP-A-11-209379.
[0062]
The most preferred procedure for pyrophosphate determination is that the sample containing pyrophosphate contains xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and inosinic acid (xanthylic acid, Acid may be added at the same time to remove hypoxanthine and / or xanthine present in the sample, and then hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) and chemiluminescent reagent are added at the same time, In this method, acid is converted into hypoxanthine and / or xanthine, and further converted into light and uric acid. Even if this operation method is used, the steps of adding the reagents need only be performed twice, which is efficient.
[0063]
For example, 2-methyl-6- (4-methoxyphenyl) -3,7-dihydroimidazo [1,2-a] pyrazin-3-one hydrochloride (MCLA), which is a kind of Cypridina luciferin derivative, is used to produce pyrroline. When measuring acid, usually about 5 to 500 mM Tris-HCl buffer containing about 1 to 100 mM inosinic acid, about 1 to 100 mM magnesium chloride, and about 1 to 100 unit / L xanthine dehydrogenase / oxidase ( After adding 20 μL to 100 μL of a sample containing pyrophosphate and reacting at 27 to 65 ° C. for about 1 to 10 minutes, MCLA of about 0.1 to 100 μM, magnesium chloride of about 1 to 100 mM, Add 80 μL of about 5 to 500 mM Tris-HCl buffer (pH 6.5 to 8.5) containing about 1000 unit / mL hypoxanthine guanine phosphoribosyltransferase, and react at 27 to 65 degrees for about 10 to 120 minutes. It may be the chemiluminescence intensity measurement.
[0064]
Since this method has a high detection limit for the pyrophosphate measurement method using a color reaction in formazan, it was proposed to increase the detection sensitivity of pyrophosphate by using chemiluminescence detection when the detection limit is low. I put it out.
[0065]
Next, the third method according to the present invention will be described. The third method is similar to the first and second methods described above, in which a necessary amount of xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) is added to the measurement sample containing pyrophosphate, Hypoxanthine and / or xanthine previously mixed therein is removed. The required amount of inosinic acid (which may contain xanthylic acid or xanthylic acid alone) and hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) are then added to reduce the pyrophosphate in the sample to hypoxanthine and / or Or change to xanthine. Xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and superoxide anion (O₂ ^-) And a fluorescent reagent that shows fluorescence is added to convert hypoxanthine and / or xanthine derived from pyrophosphate into a fluorescent substance and uric acid. This series of reactions is shown as follows.
[0066]
[Chemical 3]

[0067]
When xanthine oxidase acts on hypoxanthine, oxygen once becomes superoxide anion (O₂ ^-) Immediately reacts with water to generate hydrogen peroxide. However, superoxide anion (O₂ ^-) If there is a fluorescent reagent for detection, superoxide anion (O₂ ^-) Reacts with a fluorescent reagent to show fluorescence. As a fluorescent reagent, superoxide anion (O₂ ^-) Is preferable, and it is important that the compound does not react with hydrogen peroxide. Suitable fluorescent reagents for this purpose include Amplex^TM Examples include fluorescent reagents disclosed in Red (Anal. Biochem., 253, 162-168 (1997) or J. Immunol. Methods 202, 133-141 (1997)).
[0068]
The most preferred procedure for pyrophosphate determination is that the sample containing pyrophosphate contains xanthine dehydrogenase (EC 1.1.1.204) / xanthine oxidase (EC 1.1.3.22) and inosinic acid (xanthylic acid, Acid may be added at the same time to remove hypoxanthine and / or xanthine mixed in the sample, and then hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) and a fluorescent reagent are added at the same time to add pyrophosphate. Is converted into hypoxanthine and / or xanthine, and further converted into a fluorescent compound and uric acid. Even if this operation method is used, the steps of adding the reagents need only be performed twice, which is efficient.
[0069]
For example, Amplex^TM When measuring pyrophosphate using Red, it is usually about 5 to 500 mM containing about 1 to 100 mM inosinic acid, about 1 to 100 mM magnesium chloride, and about 1 to 100 unit / L xanthine dehydrogenase / oxidase. After adding 20 μL of Tris-HCl buffer (pH 6.5 to 8.5) to 100 μL of sample containing pyrophosphate and reacting at 27 to 65 ° C. for about 1 to 10 minutes, about 0.1 to 1000 μM of Amplex^TM Add Red, about 1 to 100 mM magnesium chloride, about 10 to 1000 unit / mL hypoxanthine guanine phosphoribosyltransferase, about 5 to 500 mM Tris-HCl buffer (pH 6.5 to 8.5), add 80 μL, and add 27 to 65 degrees. Then, the fluorescence intensity of the reaction solution (excitation wavelength: 550 to 570 nm, fluorescence wavelength: 580 to 610 nm) may be measured.
[0070]
Since this method has a high detection limit for a pyrophosphate measurement method using a color reaction in formazan, it was devised for the purpose of increasing the detection sensitivity of pyrophosphate by using fluorescence detection at a low detection limit. .
[0071]
Although the pyrophosphate measurement method of the present invention can be carried out using necessary reagents and materials obtained from separate sources, the reagents and enzymes useful for the measurement method of the present invention are one of the reagent kits. It is advantageous to be supplied as a part. The minimum amounts of enzymes, cofactors, buffers, salts, and appropriate ranges for each measurement used in the present invention are well known in the art and are included in the reagent kit provided by the present invention. The components to be produced are preferably provided and used in any concentration suitable for a given method and are pre-dispensed in a capped container. The component contained in the reagent kit of the present invention may be one containing each of the reagent and enzyme required according to the measurement sample and measurement method, or a mixture of two or more types of reagents or enzymes. . Moreover, the components contained in the reagent kit of the present invention can be added after being added or mixed separately to the sample. These materials can be appropriately packaged (dry or wet) for the purpose of necessary stability, safety and ease of handling. Alternatively, a reagent kit including a reagent support such as a filter paper on which necessary reagents are fixed as a component can also be used significantly in the measurement method of the present invention, and the above reaction can be performed on the reagent support. Is possible. As a means of detecting pyrophosphate using this reagent kit, in addition to precise quantification using advanced optical instruments such as an ultraviolet-visible spectrophotometer, a fluorescence spectrophotometer, and a chemiluminescence detector, the presence or absence of pyrophosphate As a simple determination for confirming the color density, it is also possible to know the approximate density by comparing the density with a color sample by the naked eye, or by coloring with a photograph or a CCD camera, or the density of fluorescence or light emission.
[0072]
Moreover, it is advantageous that the reagent and enzyme used when detecting or quantifying the presence or absence of nucleic acid using the pyrophosphate measurement method of the present invention are supplied as components of a reagent kit. As a component of such a reagent kit, in addition to the components included in the reagent kit for measuring pyrophosphate, a reagent necessary for amplifying nucleic acid is further included. As reagents necessary for amplifying nucleic acids, primers, cofactors, nucleotide-3-phosphates and the like can be mentioned. The minimum amount of primers, cofactors, nucleotide-3-phosphates, and appropriate ranges for each used in nucleic acid amplification are well known in the art, and these may be used at any concentration suitable for a given method. Provided and used.
[0073]
In addition to the PCR reaction, there are several methods such as the LAMP method and the ICAN method as the nucleic acid amplification method. However, when pyrophosphate is produced simultaneously with nucleic acid amplification, the method of the present invention is a method other than PCR. The same applies to the above. In the determination by dye generation, the presence or absence of a specific nucleic acid can be visually determined by adding the above-mentioned reagent useful for the pyrophosphate measurement method of the present invention to an existing nucleic acid amplification reagent without using a detection device.
[0074]
When it is desired to detect the presence or absence of a nucleic acid amplification reaction by end point analysis, a step of removing hypoxanthine and / or xanthine previously contained in the sample (the most preferred measurement method is a reagent that includes two steps in the above-described operation procedure). The first addition step) may be performed before or after the start of any of the nucleic acid amplification reactions described above. In that case, by using a heat-resistant xanthine dehydrogenase / oxidase that does not lose enzyme activity even when a temperature of 95 ° C. or higher is applied in the nucleic acid amplification reaction, a step after the removal step for pyrophosphate detection (addition step over two steps) In that case, it is not necessary to add xanthine dehydrogenase / oxidase again in the second addition step). When it is desired to detect the presence or absence of a nucleic acid amplification reaction by real-time analysis, it is necessary that both the xanthine dehydrogenase / oxidase and the hypoxanthine guanine phosphoribosyltransferase among the pyrophosphate detection reagents are thermostable.
[0075]
The PCR reaction reacts as follows to release pyrophosphate as a byproduct. Therefore, the amount of pyrophosphate (PPi) produced is proportional to the amount of amplified DNA.
[0076]
[Formula 4]

[0077]
When the amount of initial template nucleic acid such as a virus is quantified according to the present invention, the fact that pyrophosphate is generated exponentially by PCR is used. The PCR reaction product shows an approximately exponential increase at the beginning of the reaction, and when a sufficient number of cycles of PCR is performed, almost the same amount of PCR reaction product is obtained regardless of the initial template nucleic acid amount. Therefore, the number of cycles (CT) at which a PCR reaction product reaches a certain amount is inversely related to the initial template nucleic acid amount. In order for this relationship to be established, the amplification factor in the exponential amplification period must be the same even if the amount of the initial template nucleic acid is different. Since this relationship is established when the target sequences are the same, the initial template nucleic acid amount can be estimated from CT. In the present invention, a method is used in which PCR reaction is carried out while changing the number of cycles, and the amount of initial template nucleic acid is determined from the amount of pyrophosphate produced in each number of cycles. For example, the amount of initial template nucleic acid can be quantified from the approximate pyrroline concentration and the change over time by analyzing the change over time of color data using a personal computer.
[0078]
【Example】
The present invention will now be described in more detail by way of examples, which are presented for the purpose of illustration and are not intended to limit the invention.
[0079]
Example 1
First, the following two types of reagents are prepared in advance.
[0080]
(1) 100 mM Tris-HCl buffer (pH 7.6) containing 20 mM inosinic acid, 10 mM magnesium chloride, 0.4 units / mL xanthine dehydrogenase / oxidase
(2) 100 mM Tris-HCl buffer (pH 7.6) containing 4 mM paraiodonitrotetrazolium violet, 10 mM magnesium chloride, 100 units / mL hypoxanthine guanine phosphoribosyltransferase
20 μl of (1) was added to 100 μl of a pyrophosphoric acid solution having a known concentration and reacted at 60 ° C. for 1 minute, and then 80 μl of (2) was added and reacted at 60 ° C. for 10 minutes. 90 μl of this reaction solution was placed in a 96-well microplate, and absorbance at 515 nm and 630 nm was measured using a microplate reader “Bio-Tek Elx 800” (manufactured by Bio-Tek Instruments). The calibration curve of pyrophosphate at that time is shown in FIG. The absorbance in FIG. 1 was determined by subtracting the absorbance at 630 nm from the absorbance at 515 nm. From the results of this example, it can be seen that pyrophosphate can be measured with high accuracy using the method of the present invention.
[0081]
(Example 2)
Reagents similar to Example 1 were prepared and 10% and 50% bovine serum or 1-10 mM potassium phosphate (KH₂PO_Four), 20 μl of (1) is added to 100 μl of 10 mM Tris HCl solution (pH 7.8) containing 500 μM pyrophosphate containing 1-10 mM ATP, 50-500 mM hypoxanthine, 50-500 mM xanthine, After reacting at 1 ° C. for 1 minute, 80 μl of (2) was added and reacted at 60 ° C. for 10 minutes. 90 μl of this reaction solution was placed in a 96-well microplate, and absorbance at 515 nm and 630 nm was measured using a microplate reader “Bio-Tek Elx 800” (manufactured by Bio-Tek Instruments). The absorbance at that time is shown in Table 1. The absorbance in Table 1 was determined by subtracting the absorbance at 630 nm from the absorbance at 515 nm. From the results of this Example, it can be seen that when the method of the present invention is used, pyrophosphate can be measured without being affected by phosphoric acid and ATP present in biological samples and various biological samples. Further, hypoxanthine and xanthine, which are background causative substances, did not affect the measured value even when about 50 to 100 mM was present in the sample. When measuring samples containing hypoxanthine or xanthine of 100 mM or more, (1) increase the concentration of xanthine dehydrogenase / oxidase in the solution, or (1) increase the reaction time after adding the solution. It is clear that this should be done.
[0082]
[Table 1]

[0083]
(Example 3)
Nucleic acids were amplified by PCR using Amersham Pharmacia Biotech Co., Ltd. Ready-To-Go PCR Breads. A reaction solution for PCR was prepared using Control Lambda DNA attached to the kit and a set of primers according to the protocol attached to the kit. Then, cycles of denaturation (95 ° C., 1 minute), annealing (55 ° C., 1 minute), and DNA extension reaction (72 ° C., 1 minute) were performed for 0, 10, 20, 25, 27, 30, 35 cycles. . The composition of the reaction solution is as follows.
[0084]
Composition of reaction solution (in one PCR Breads bead)
Control Lambda DNA 1 μl (0 or 1 ng / μl)
Control Primer 1 1μl
Control Primer 2 1μl
Sterile distilled water 22 μl
After completion of PCR, colorimetric quantification was performed on 50 μl of the reaction solution using the same method as in Example 1. The relationship between the absorbance and the number of cycles at that time is shown in FIG. From this figure, it can be seen that the absorbance increases as the number of cycles increases, so that the presence or absence of the target nucleic acid can be determined using this measurement method.
[0085]
Example 4
First, the following two types of reagents are prepared in advance.
[0086]
(1) 100 mM Tris-HCl buffer (pH 7.6) containing 20 mM inosinic acid, 10 mM magnesium chloride, 0.4 units / mL xanthine dehydrogenase / oxidase
(2) 100 mM Tris-HCl buffer (pH 7.6) containing 32 μM MCLA, 10 mM magnesium chloride, 100 units / mL hypoxanthine guanine phosphoribosyltransferase
Take 50 μl of pyrophosphoric acid solution of known concentration in a microtube, add 10 μl of (1) and react at 60 ° C. for 1 minute, then add 40 μl of (2) and chemiluminescence for 10 minutes while reacting at 60 ° C. The intensity (chemiluminescence detector, Hamamatsu Photonics) was measured. The calibration curve of pyrophosphate at that time is shown in FIG. From the results of this example, it can be seen that pyrophosphate can be measured with high accuracy using the method of the present invention.
[0087]
(Example 5)
Nucleic acids were amplified by PCR using Amersham Pharmacia Biotech Co., Ltd. Ready-To-Go PCR Breads. The reaction solution for PCR was prepared using Control Lambda DNA attached to the kit (0, 0.1, 1, 10 ng / μl) and a set of primers according to the protocol attached to the kit. Then, cycles of denaturation (95 ° C., 1 minute), annealing (55 ° C., 1 minute), and DNA extension reaction (72 ° C., 1 minute) were performed 0, 10, 15, 20, 25, and 27 cycles. The composition of the reaction solution is as follows.
[0088]
Composition of reaction solution (in one PCR Breads bead)
Control Lambda DNA 1μl (prepared to 4 different concentrations)
Control Primer 1 1μl
Control Primer 2 1μl
Sterile distilled water 22 μl
After the completion of PCR, the chemiluminescence intensity was measured for 25 μl of the reaction solution using the same method as in Example 4. The relationship between the chemiluminescence intensity and the number of cycles at that time is shown in FIG. From this figure, it can be seen that the chemiluminescence intensity increases with an increase in the number of cycles, so that it is possible to determine the presence or absence of the target nucleic acid and estimate the initial template nucleic acid amount using this measurement method.
[0089]
(Example 6)
First, the following two types of reagents are prepared in advance.
[0090]
(1) 100 mM Tris-HCl buffer (pH 7.6) containing 20 mM inosinic acid, 10 mM magnesium chloride, 0.4 units / mL xanthine dehydrogenase / oxidase
(2) 200μM Amplex^TM Red, 10 mM magnesium chloride, 100 mM Tris-HCl buffer (pH 7.6) containing 100 units / mL hypoxanthine guanine phosphoribosyltransferase
Take 50 μl of pyrophosphoric acid solution of known concentration in a microtube, add 10 μl of (1) and react at 60 ° C. for 1 minute, add 40 μl of (2), react at 60 ° C. for 10 minutes, and then fluoresce The intensity (fluorescence spectrophotometer / JASCO) was measured. The calibration curve of pyrophosphate at that time is shown in FIG. From the results of this example, it can be seen that pyrophosphate can be measured with high accuracy using the method of the present invention.
[0091]
(Example 7)
The cultured Salmonella Tonpson, Salmonella Enteritidis, Escherichia coli O6 and O148 are extracted with hot water, and then PCR reaction is performed using a Salmonella invA gene detection primer (Takara Bio). As a result of detection, only S. Tonpson and S. Enteritidis were colored (Table 2). The amount of pyrophosphate in the reaction solution in which the generation of the PCR reaction product was confirmed by agarose gel electrophoresis was about 200-400 μM. Thus, by using this method, bacteria could be identified by using a non-toxic reagent and by simple visual judgment.
[0092]
[Table 2]

[0093]
【The invention's effect】
As described above in detail, the present invention is a simple and accurate pyrophosphate quantification method that overcomes the background increase, which is a drawback of the conventional pyrophosphate quantification method, and a reagent kit useful for the quantification method. The invention makes it possible to accurately and easily determine the presence or absence of a specific nucleic acid.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between pyrophosphate concentration and absorbance obtained by using the pyrophosphate quantification method of the present invention.
FIG. 2 is a graph showing the relationship between the number of cycles and the absorbance obtained by using the pyrophosphate quantification method of the present invention.
FIG. 3 is a graph showing the relationship between pyrophosphate concentration and chemiluminescence intensity obtained by using the pyrophosphate determination method of the present invention.
FIG. 4 is a graph showing the relationship between the number of cycles and chemiluminescence intensity obtained using the pyrophosphate quantification method of the present invention.
FIG. 5 is a graph showing the relationship between the pyrophosphate concentration and the fluorescence intensity obtained using the pyrophosphate quantification method of the present invention.

Claims (14)

  A pyrolin comprising the steps of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and adding a tetrazolium salt and hypoxanthine guanine phosphoribosyltransferase Acid detection and quantification method.   A step of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and a step of adding a chemiluminescent reagent for detecting a superoxide anion and hypoxanthine guanine phosphoribosyltransferase A method for detecting and quantifying pyrophosphate.   Adding a step of adding inosinic acid and / or xanthylic acid and xanthine dehydrogenase / oxidase to a solution containing pyrophosphate, and a step of adding a fluorescent reagent for detecting a superoxide anion and hypoxanthating anine phosphoribosyltransferase. A method for detecting and quantifying pyrophosphate. A method for detecting and quantifying nucleic acid, comprising the step of amplifying nucleic acid in a sample solution and pyrophosphoric acid contained in the sample solution after amplification using the method according to any one of claims 1 to 3. A method for detecting and quantifying nucleic acid, comprising a step of detecting and quantifying the sample. Characterized by using the hypoxanthine guanine phosphoribosyl transferase and / or xanthine dehydrogenase / oxidase be added to temperatures above 95 ° C. without loss of enzymatic activity, pyrroline according to any one of claims 1 to 3 Acid detection and quantification method. 5. The method for detecting and quantifying nucleic acid according to claim 4 , wherein hypoxanthine guanine phosphoribosyl and / or xanthine dehydrogenase / oxidase that does not lose enzyme activity even when a temperature of 95 ° C. or higher is applied. A reagent kit useful for the pyrophosphate detection / quantification method according to claim 1 , comprising pyrophosphate and inosinic acid and / or xanthylic acid, tetrazolium salt, hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase, A reagent kit containing each as a reagent contained alone or as a mixed reagent of any two or more. A reagent kit useful for the pyrophosphate detection / quantification method according to claim 2 , comprising a chemiluminescence reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, and hypoxanthine guanine phosphoribosyltransferase. A reagent kit containing xanthine dehydrogenase / oxidase as a reagent containing each singly or as a mixed reagent of any two or more thereof. A reagent kit useful for the pyrophosphoric acid detection / quantification method according to claim 3 , comprising a fluorescent reagent, hypoxanthine guanine phosphoribosyltransferase and xanthine for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion. A reagent kit comprising dehydrogenase / oxidase as a reagent containing each alone or as a mixed reagent of any two or more. A reagent kit useful for detection and quantification of a target nucleic acid, comprising a reagent kit necessary for amplifying a nucleic acid in addition to the reagent kit according to any one of claims 7 to 9 . A reagent kit useful for the pyrophosphate detection / quantification method according to claim 1 , wherein pyrophosphate and inosinic acid and / or xanthylic acid, tetrazolium salt, hypoxanthine guanine phosphoribosyltransferase and xanthine dehydrogenase / oxidase are immobilized. A reagent kit comprising the prepared reagent support. A reagent kit useful for the pyrophosphate detection / quantification method according to claim 2 , comprising a chemiluminescence reagent for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion, and hypoxanthine guanine phosphoribosyltransferase. A reagent kit comprising a reagent support on which xanthine dehydrogenase / oxidase is immobilized. A reagent kit useful for the pyrophosphoric acid detection / quantification method according to claim 3 , comprising a fluorescent reagent, hypoxanthine guanine phosphoribosyltransferase and xanthine for detecting pyrophosphate and inosinic acid and / or xanthylic acid and superoxide anion. A reagent kit comprising a reagent support on which dehydrogenase / oxidase is immobilized. The reagent kit according to any one of claims 11 to 13 , wherein a reagent necessary for further amplifying the nucleic acid is fixed to the reagent support.

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