JP5178528B2 - 分子診断用増幅システムおよび方法 - Google Patents
分子診断用増幅システムおよび方法 Download PDFInfo
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- JP5178528B2 JP5178528B2 JP2008548567A JP2008548567A JP5178528B2 JP 5178528 B2 JP5178528 B2 JP 5178528B2 JP 2008548567 A JP2008548567 A JP 2008548567A JP 2008548567 A JP2008548567 A JP 2008548567A JP 5178528 B2 JP5178528 B2 JP 5178528B2
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Description
本発明は、温度サイクル法および等温法に基づいて増幅を実施することができる、一体型の核酸試験カートリッジに関する。さらに、本発明は、核酸標的を含むと推測される試料を受け取り、増幅を実施し、かつ検出用にアンプリコンを移送するための装置および方法に関する。増幅カートリッジは、少なくとも光学的センサーおよび電気化学的センサーを含む検知手段を装備していてよい。カートリッジは、限定されるわけではないが、ポリメラーゼ連鎖反応、ローリングサークル増幅、および鎖置換増幅を含む、様々な増幅方法を実施することができる。増幅装置はまた、持ち運び可能な電源またはそのための手段を用いて機能する能力も有する。
核酸試験の用途は幅広い。従来の商業的試験の大多数は、クラミジア、淋病、肝炎、およびヒト免疫不全ウイルス(HIV)ウイルス負荷を含む感染症;嚢胞性線維症を含む遺伝的疾患;凝血およびヘモクロマトーシスを含む血液学因子;ならびに乳癌遺伝子を含む癌に関する。対象となる他の分野には、法医学および父子鑑別、薬理ゲノミクスと呼ばれる心血管疾患および薬物耐性のスクリーニングが含まれる。試験の大多数は、現在、持ち運び不可能かつ操作の複雑な機器を用いて、集約された実験室において行われる。慣例的に、試験は通常、アッセイ法を実施するために高度な熟練者を必要とする。結果として、特定の核酸断片を含むと推測される試料を得てから、その有無を決定するまでの間にかかる時間が、しばしば数時間、さらには数日にさえなる。しかしながら、他の種類の血液試験と同様に、医師および科学者は、しばしば、簡便でユーザーフレンドリーな様式で得られる結果をより迅速に必要とする。その結果として、核酸試験を迅速かつ簡便に実施することができる持ち運び可能な解析システムが必要とされている。
本発明の目的は、増幅をすることができる一体型の核酸試験カートリッジを提供することである。
本発明の例示的な態様によれば、図2の核酸増幅カートリッジ10は、単回使用で、かつ低コストであるように設計されている。さらに、これはまた、使用される試薬および患者の生物試料を装置内に安全に保持する様式で、使い捨てである。この装置は、簡便な様式でアンプリコンを作製することができ、かつ、実験室の外の治療現場位置で使用することさえできる。カートリッジ装置は、可逆的シール13を有する入口12、可逆的シール15を有する出口14、およびまた、密閉可能な試料投入口16を有する増幅チャンバー11を含むハウジングを含む。増幅カートリッジ10は、熱伝導性材料、好ましくはシリコンなどで作られた、チャンバー11の一部分を形成する壁17を含む。あるいは、壁17は、アルミナ、石英、ガリウムヒ素、および熱伝導性プラスチックなどで作られていてもよい。壁17は、内表面18および外表面19(図7(c)を参照されたい)を含み、かつ、外表面19上には、加熱回路20(図7(c)を参照されたい)および温度センサー21がある。これらの構成要素は、例えば、金属がシリコンウェハ表面にパターン焼き付けされる周知の微細加工技術によって、導電性インクのスクリーン印刷によって、または他の同様の技術によってなど、任意で、壁表面に直接的に製作される。ウェハが使用される場合、それは、個々のチップに切り離され(diced)てよく、かつ、増幅チャンバー11を形成するために第2のプラスチック構成要素22との組立ておよび接着によって壁を形成するのに使用され得る。試料投入口16により、核酸試料が増幅用のチャンバー11に導入されることが可能になる。
ヘマクロマトーシス(Hfe)C282Y対立遺伝子のPCR増幅および検出
記号表示:5Bio-5'-ビオチン化塩基、iBiodT-内部dTビオチン化塩基、*-ホスホロチオラート主鎖、T20-配列中の20dT、アミノ修飾因子_C12-5'アミノ誘導体、iSpC3-スペーサー/遮断物ホスホルアミダイト、Hfe-ヘマクロマトーシス遺伝子、Wt-野生型、Mut-変異体、SNP-一塩基多型、MBW-選択された配列、Sc-選択された配列。
フェニルチオカルバミド(PTC)対立遺伝子1のPCR増幅および検出[TAS2R38、Ala49Pro]
記号表示:5Bio-5'-ビオチン化塩基、iBiodT-内部dTビオチン化塩基、*-ホスホロチオラート主鎖、T20-配列中の20dT、アミノ修飾因子_C12-5'アミノ誘導体、PTC-フェニルチオカルバミド遺伝子、Wt-野生型、Mut-変異体、SNP-一塩基多型、MBW-選択された配列、Sc-選択された配列。
Claims (38)
- ハウジングと、
第1の可逆的シールを有する入口、
第2の可逆的シールを有する出口、
密閉可能な試料投入口、および
増幅チャンバーの一部分を形成する第1の壁
を含む、増幅チャンバーと
を含む、アンプリコンを作製するための単回使用核酸増幅装置であって、
該第1の壁が、熱伝導性材料を含み、かつ第1の表面および第2の表面を含み、
該第2の表面が、加熱回路および温度センサーを含み、
該試料投入口により、核酸試料が該増幅チャンバーに入ることが可能になり、
該入口が、ポンプおよび貯蔵器と共に、第1の導管に連結されており、
該出口が、該増幅チャンバーからアンプリコンが出て行くのを可能にする第2の導管に連結されており、
前記ポンプが、第1の可撓性の隔膜を含み、
前記第1の可逆的シールが、第2の可撓性の隔膜を含み、かつ
前記第2の可逆的シールが、第3の可撓性の隔膜を含む、
単回使用核酸増幅装置。 - 第1の可撓性の隔膜が、前記増幅装置に係合して作動させるための制御機器上のプランジャーと係合し、かつそれによって作動され得る、請求項1記載の増幅装置。
- ポンプが空気ポンプを含む、請求項1記載の増幅装置。
- 貯蔵器が液体袋を含む、請求項1記載の増幅装置。
- 液体袋が、核酸増幅を実施するための液体を含む、請求項4記載の増幅装置。
- 貯蔵器が第4の可撓性の隔膜を含む、請求項1記載の増幅装置。
- 第4の可撓性の隔膜が、前記増幅装置に係合して作動させるための制御機器上のプランジャーと係合し、かつそれによって作動され得る、請求項6記載の増幅装置。
- 第1の壁がシリコンを含む、請求項1記載の増幅装置。
- シリコンが、増幅チャンバーの第1の表面の面積の約30〜約50パーセントを含む、請求項8記載の増幅装置。
- 増幅チャンバーが、プラスチック材料を含む第2の壁を含む、請求項1記載の増幅装置。
- 第2の壁が、約0.2mm〜約5mmの範囲の壁厚を含み、かつ第2の壁が、1つまたは複数の付加的なリブ支持体を含む、請求項10記載の増幅装置。
- 増幅チャンバーの内容積が、約5μL〜約50μLの範囲である、請求項1記載の増幅装置。
- 増幅チャンバー容積に対する増幅チャンバー表面の比率が、約5〜約30立方mmの増幅チャンバー容積に対して、約50〜約200平方mmの範囲の増幅チャンバー表面の範囲である、請求項1記載の増幅装置。
- 増幅チャンバーの内部形状が、長方形の構造、角の丸い長方形の形状、円柱、および断面が楕円形である円柱状の構造の内の1つを含む、請求項1記載の増幅装置。
- 第1の壁の第2の表面が加熱回路を含む、請求項1記載の増幅装置。
- 加熱回路が、第2の表面に作られた抵抗性の電気的通路を、該通路を通る電流を提供するための外部回路と接触させるための連結パッドと共に含む、請求項15記載の増幅装置。
- 第1の壁の第2の表面が温度センサーを含む、請求項1記載の増幅装置。
- 温度センサーが、第2の表面に組み立てられたサーミスターおよび熱電対の内の1つを、該サーミスターおよび該熱電対の内の1つに連結するための外部回路と接触させるための連結パッドと共に含む、請求項17記載の増幅装置。
- 試料投入口が、試料導入エレメントと結合することができる、請求項1記載の増幅装置。
- 試料導入エレメントが、
核酸試料を回収および保持することができる吸収性パッドを有する第1の端部と、
ハンドルを形成する第2の端部と
を含むワンドを含み、
該第1の端部が、試料投入口を経て増幅チャンバーへと通過することができ、かつ
該ワンドが、試料投入口に該ワンドを係合しかつ密閉するために、該第1の端部と該第2の端部との間に係合構造を含む、
請求項19記載の増幅装置。 - 係合構造が、ワンド上の雄ネジ構造および試料投入口上の雌ネジ構造を含む、請求項20記載の増幅装置。
- 係合構造が、ワンド上の雄型環状ロック構造および試料投入口上の雌型環状ロック構造を含む、請求項20記載の増幅装置。
- 増幅チャンバーが、第1の壁の第1の表面の少なくとも一部分上に、少なくとも1つの糖ならびに少なくとも1つの脱水およびガラス化された試薬を含むコーティングを含む、請求項1記載の増幅装置。
- 増幅チャンバーが、約10〜約50℃/秒の範囲の温度上昇変化速度が可能なものである、請求項1記載の増幅装置。
- 増幅チャンバーが、約4〜約50℃/秒の範囲の温度低下変化速度が可能なものである、請求項1記載の増幅装置。
- 増幅チャンバーが光学的窓を含む、請求項1記載の増幅装置。
- 第1の壁の第2の表面が、外部回路と接触させるための連結パッドを有するペルティエ回路を含む、請求項1記載の増幅装置。
- 第2の可撓性の隔膜を、加力することによって閉じた位置へ、および加力を無くすことによって開いた位置へと動かすことができる、請求項1記載の増幅装置。
- 第2の可撓性の隔膜を、第2の可撓性の隔膜と結合するピンを有する係合された機器によって提供される加力により、閉じた位置へと動かすことができる、請求項1記載の増幅装置。
- 第3の可撓性の隔膜を、加力することによって閉じた位置へ、および加力を無くすことによって開いた位置へと動かすことができる、請求項1記載の増幅装置。
- 第3の可撓性の隔膜を、第3の可撓性の隔膜と結合するピンを有する係合された機器によって提供される加力により、閉じた位置へと動かすことができる、請求項1記載の増幅装置。
- 第2の導管が、アンプリコンの検出用の装置と係合するための結合特性を含む、請求項1記載の増幅装置。
- 第1の導管が、液体検出センサーを有するチップ挿入物を含む、請求項1記載の増幅装置。
- 第1の表面が内表面を含み、かつ
第2の表面が外表面を含む、
請求項1記載の増幅装置。 - 単回使用装置中でアンプリコンを作製するための核酸増幅方法であって、
a)試料投入口を介して増幅チャンバー中に核酸試料を導入する段階、
b)開口部を密閉する段階、
c)可逆的に密閉可能な入口を介して、液体を貯蔵器から増幅チャンバーに移送する段階、
d)増幅チャンバーの入口および可逆的に密閉可能な出口を密閉する段階、
e)液体を試料と混合して、核酸、緩衝剤、ポリメラーゼ、および1つまたは複数のプライマーを含む混合物を形成させる段階、
f)第1の温度と第2の温度との間で、所定の時間および所定のサイクル回数、増幅チャンバーの温度を循環させて、アンプリコンを形成させる段階、
g)チャンバーの入口および出口を開放する段階、ならびに
h)入口にポンプで空気力を加えて、出口を介してアンプリコンをチャンバーから移動させる段階
を含み、ここで、
前記ポンプが、第1の可撓性の隔膜を含み、
前記可逆的に密閉可能な入口が、第2の可撓性の隔膜を含み、かつ
前記可逆的に密閉可能な出口が、第3の可撓性の隔膜を含む、
方法。 - 単回使用装置中でアンプリコンを作製するための核酸増幅方法であって、
a)試料投入口を介して増幅チャンバー中に核酸試料を導入する段階、
b)開口部を密閉する段階、
c)可逆的に密閉可能な入口を介して、液体を貯蔵器から増幅チャンバーに移送する段階、
d)チャンバーの入口および可逆的に密閉可能な出口を密閉する段階、
e)液体を試料と混合して、核酸、緩衝剤、ポリメラーゼ、および1つまたは複数のプライマーを含む混合物を形成させる段階、
f)所定の時間、等温増幅温度までチャンバーの温度を上昇させて、アンプリコンを形成させる段階、
g)増幅チャンバーの入口および出口を開放する段階、ならびに
h)入口にポンプで空気力を加えて、出口を介してアンプリコンをチャンバーから移動させる段階
を含み、ここで、
前記ポンプが、第1の可撓性の隔膜を含み、
前記可逆的に密閉可能な入口が、第2の可撓性の隔膜を含み、かつ
前記可逆的に密閉可能な出口が、第3の可撓性の隔膜を含む、
方法。 - 前記密閉可能な試料投入口が、ワンドを含む試料導入エレメントと結合するよう構成され、
該ワンドは、核酸試料を収集し保持するよう構成された吸収性パッドを有する第1の端部と、使用者が握るハンドルとして構成された第2の端部と、を含み、
該ワンドの第1の端部は、前記密閉可能な試料投入口を介して前記増幅チャンバーに通過するよう構成され、
該ワンドは、前記試料投入口内で該ワンドと係合し密閉するための前記第1の端部および前記第2の端部の間に係合構造を含む、
請求項1記載の増幅装置。 - 前記増幅チャンバーが、
低熱伝導特性を有する第2の壁、
前記第1の壁と該第2の壁との間のガスケット、および
前記増幅チャンバーを冷却するよう構成されたヒートシンク
をさらに含む、請求項1記載の増幅装置。
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WO2007078850A3 (en) | 2008-04-17 |
WO2007078850A2 (en) | 2007-07-12 |
JP2009521924A (ja) | 2009-06-11 |
US8703445B2 (en) | 2014-04-22 |
EP1966366A2 (en) | 2008-09-10 |
US20070154922A1 (en) | 2007-07-05 |
EP1966366A4 (en) | 2011-06-15 |
WO2007078850A9 (en) | 2007-08-30 |
CA2634735A1 (en) | 2007-07-12 |
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