JPWO2020045591A1 - Pcr反応容器 - Google Patents
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Abstract
Description
〔1〕
基板と、
前記基板に形成された流路と、
前記流路の両端に設けられた一対のフィルタと、
前記フィルタを通じて前記流路と連通する一対の空気連通口と、
前記流路における前記一対のフィルタの間に形成されたサーマルサイクル領域と、
前記流路に上から試料を注入することができる試料注入口を備え、
前記試料注入口の前記基板の表面部面積は0.7〜1.8mm2であることを特徴とする
PCR反応容器。
〔2〕
前記試料注入口が円形又は楕円形又は多角形である〔1〕記載の反応容器。
〔3〕
前記流路の幅が300〜1000μmである〔1〕又は〔2〕に記載の反応容器。
〔4〕
別途試料注入に使用する円形もしくは多角形の管状の形状を有する試料注入部材の先端が流路の内部まで到達することを特徴とする〔1〕〜〔3〕のいずれ1項に記載の反応容器。
〔5〕
さらに前記試料注入口の容積(前記基板表面から前記流路間の空間)が7.5μL以下である〔1〕〜〔4〕のいずれか1項に記載の反応容器。
〔6〕
試料注入後において前記試料注入口の上部開口部がシールまたは前記試料注入部材等により密閉されることを特徴とする〔1〕〜〔5〕のいずれか1項に記載の反応容器。
[7]
厚みが3〜5mmである〔1〕〜〔6〕のいずれか1項に記載の反応容器。
なお、空気連通口24、26とは別に空気の出口を設け、第4封止フィルムの貼り付け/剥がしにより流路内への試料注入を行なうことも可能である。
実施例1
1.使用するPCR装置には高速なサーマルサイクルが可能な様に、2カ所の温度帯の上へPCR試薬を交互に搬送するための1本の流路を有する往復送液型のPCR反応容器(厚み4mm)を利用した。
2.PCR反応容器の樹脂製の基板に形成された流路の中心軸と直行する様に、当該基板の上面から直径0.9 〜 1.6 mmのドリルを用いて貫通孔を形成し、試薬注入口を作製した。余分なバリや汚れを除去した後、流路封止フィルムを含む全ての封止フィルムを接合し、以後のPCRの検証を行った。
3.以下のようにPCR試薬を調製した。
5.マイクロピペットからPCR試薬を排出する際、通常は使い捨てピペットチップの先端位置において排出した溶液が切り取られる。そのため、試薬注入口の径との関係によりピペットチップの先端位置が、完全に流路内まで到達せず試薬注入口内に止まることが考えられる。この場合、流路中に注入されプラグ状になったPCR試薬の後端が試薬注入口内に留ることとなり、以後のPCRにおける送液時に、一部のPCR試薬が試薬注入口に残存することとなる。
6.一方、このPCR試薬の注入時に、マイクロピペットを過剰量押し込むことにより、吸引したPCR試薬の全量を排出した後、空気も引き続き流路内へ押し出すことで、PCR試薬のプラグの後端を含め完全に流路内部まで注入することが可能である。このように、マイクロピペットにより完全にPCR試薬全量を流路内まで押し込む条件を、試薬押し込み有りと表現し、一方、一般的なマイクロピペットの操作により流路内部までPCR試薬を押し込まない場合については、以後、試薬押し込み無しと表現する。
7.PCR試薬を注入し封止フィルムでシールしたPCR反応容器について、98℃および61℃の温度帯と、往復送液用のポンプと、流路内の増幅DNAを定量する蛍光検出器を内蔵した装置に装着し、リアルタイムPCRを行った。なお、PCRの条件は以下の通りとした。
1.表2に、試料注入口にピペットチップを挿入した際のピペットチップ先端の到達位置をまとめた。
4.このように、ピペットチップが試薬注入口に入らない、もしくはピペットチップ先端に比べ大きな穴径の場合は注入ができずにPCR試薬が溢れることがわかった。
5.試薬注入口の直径が1.5 mmの場合にはピペットチップの先端が流路内部まで到達していたが、試薬の押し込み無しの場合において、PCR試薬プラグの後端が試薬注入口に入り込んでいた。これは試薬注入後にピペットチップを引き抜く際に引き戻されたためと考えられる。
6.次に、リアルタイムPCRを行った結果の増幅曲線を図4に示す。
7.Ct値で2サイクル程度の差異については測定装置由来の不確かさの範囲であり、有意な差は確認されなかった。
8.以上の結果から、試薬注入口の形成に用いたドリルのサイズ毎の試薬の注入とPCRが可能かの評価を表4にまとめた。
10.ただし、正常にPCR試薬を注入できた何れの条件においてもPCRの送液には影響せず図4の通り正常にリアルタイムPCRが可能であった。
11.なお、PCR後に試料注入口に残留した液量を表5にまとめた。試薬注入口に残った試薬量はPCR前後でほぼ変化が見られなかった。
12 流路
14 基板
16 流路封止フィルム
18 第1封止フィルム
20 第2封止フィルム
22 第3封止フィルム
24 第1空気連通口
26 第2空気連通口
28 第1フィルタ
30 第2フィルタ
133 試料注入口
Claims (7)
- 基板と、
前記基板に形成された流路と、
前記流路の両端に設けられた一対のフィルタと、
前記フィルタを通じて前記流路と連通する一対の空気連通口と、
前記流路における前記一対のフィルタの間に形成されたサーマルサイクル領域と、
前記流路に上から試料を注入することができる試料注入口を備え、
前記試料注入口の前記基板の表面部面積は0.7〜1.8mm2であることを特徴とする
PCR反応容器。 - 前記試料注入口が円形又は楕円形又は多角形である請求項1記載の反応容器。
- 前記流路の幅が300〜1000μmである請求項1又は2に記載の反応容器。
- 別途試料注入に使用する円形もしくは多角形の管状の形状を有する試料注入部材の先端が流路の内部まで到達することを特徴とする請求項1〜3のいずれ1項に記載の反応容器。
- さらに前記試料注入口の容積(前記基板表面から前記流路間の空間)が7.5μL以下である請求項1〜4のいずれか1項に記載の反応容器。
- 試料注入後において前記試料注入口の上部開口部がシールまたは前記試料注入部材等により密閉されることを特徴とする請求項1〜5のいずれか1項に記載の反応容器。
- 厚みが3〜5mmである請求項1〜6のいずれか1項に記載の反応容器。
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