JP4633730B2 - 可動検出モジュールを用いる蛍光検出システム及び方法 - Google Patents
可動検出モジュールを用いる蛍光検出システム及び方法 Download PDFInfo
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- JP4633730B2 JP4633730B2 JP2006532916A JP2006532916A JP4633730B2 JP 4633730 B2 JP4633730 B2 JP 4633730B2 JP 2006532916 A JP2006532916 A JP 2006532916A JP 2006532916 A JP2006532916 A JP 2006532916A JP 4633730 B2 JP4633730 B2 JP 4633730B2
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Description
図1は、本発明の実施形態によるサーマルサイクル装置100の概略図である。装置100は、ベースユニット110と蓋アセンブリ112から成る。ベースユニット110は、従来の設計とすることができ、プログラマブルプロセッサ及びクロックなどのような従来の電子構成要素(図示せず)を通じてサーマルサイクル処理のための電力と制御機能を提供する。ベースユニット110は、キーパッド118とLCDディスプレイスクリーン120を含むことができ、ユーザがサーマルサイクラの作動を制御してモニタすることができるユーザインタフェース116もまた提供する。ベースユニット110は、電源ケーブル121を通じて外部電源(例えば、標準の120V交流電源)に接続される。ベースユニット110のいくつかの例は、本発明の譲受人であるMJ・リサーチ・インコーポレーテッドから販売されている「DNAエンジン(登録商標)」、「Dyad(登録商標)」、及び「Tetrad(登録商標)」サーマルサイクラを含む。
本明細書に説明する装置は、蛍光量を検出することによって、増幅反応で生成された増幅生成物の量を検出するために使用することができる。DNA又はRNAサンプル中に存在するターゲット配列を定量化するために、様々な増幅技術を使用することができる。増幅される配列に対して相補的な配列を含む核酸アンプリコン(コピー)の酵素的合成を伴うそのような技術は、当業技術では公知であり、広く使用されている。これらは、以下に限定はしないが、ポリメラーゼ連鎖反応(PCR)、RT−PCR、鎖置換増幅(SDA)、転写ベースの増幅反応、リガーゼ鎖反応(LCR)、及びその他(例えば、Dieffenfach及びDveksler著「PCRプライマー:実験用マニュアル」、1995年;米国特許第4,683,195号及び第4,683,202号、Innis他編集「PCRプロトコル:方法及び応用のためのガイド」、1990年;Walker他、「核酸研究」、20(7)、1691−6、1992年;Walker、「PCR方法応用」、3(1):1−6、1993年;Phyffer他、「J.Clin.Microbiol.」、34:834−841、1996年;Vuorinen他、「J.Clin.Microbiol.」、33:1856−1859、1995年;Compton、「Nature」、350(6313):91−2、1991年;Lisby、「Mol.Biotechnol.」、12(1):75−99、1999年;Hatch他、「Genet.Anal.」、15(2):35−40、1999年;及びIqbal他、「Mol.Cell Probes」、13(4):315−320、1999年を参照されたい)を含む。核酸増幅は、サンプル中に存在するターゲット配列の量が非常に少ない時に特に有益である。関連の有機体又はウイルスに属するサンプル中の核酸の検出をより確実にするのに検定の始めで必要なターゲット配列のコピーがより少ないので、ターゲット配列を増幅して合成されたアンプリコンを検出することにより、検定の感度を大幅に改善することができる。
蛍光プローブを利用するいくつかのフォーマットが利用可能である。これらのフォーマットは、多くの場合、蛍光共鳴エネルギ移動(FRET)に基づいており、分子標識と「TaqMan(登録商標)」プローブを含む。FRETは、供与体及び受容体分子間の距離依存型相互作用である。供与体及び受容体分子は、燐光体である。燐光体が部分的に重なる励起及び放射スペクトルを有する場合、非常に近傍で(通常、約10−100オングストローム)、供与体燐光体の励起は、受容体燐光体に転送される。その結果、供与体分子の寿命が短縮されてその燐光体は消失し、一方で受容体分子の燐光体輝度は、増強されて消極される。供与体の励起状態エネルギが非燐光受容体に転送される時に、供与体の蛍光は、受容体による蛍光のその後の放射なしに消失する。この場合、受容体は、消失試薬として機能する。
例示的なPCR反応条件は、一般的に、2又は3ステップサイクルを含む。2ステップサイクルは、変成ステップを有し、混成化/伸長ステップへと続く。3ステップサイクルは、変成ステップを有し、混成化ステップに続き、別の伸長ステップへと続く。ポリメラーゼ反応は、プライマーがターゲット配列に混成化されてポリメラーゼによって伸張される条件下で培養される。増幅の反応サイクル条件は、プライマーが特にターゲット配列に混成化されて伸張されるように選択される。
図8は、装置100を使用する核酸増幅と測定処理800のフローチャートである。この例では、装置100は、PCR増幅処理を制御し、核酸サンプル中の複数のターゲット配列の存在を検出する。
本発明を特定的な実施形態に関連して説明したが、多くの修正が可能であることを当業者は認識するであろう。例えば、本明細書に説明した蛍光検出アセンブリは、広範囲のサーマルサイクラシステムと共に使用するように適応させることができ、特定機器の設計に応じてあらゆる方向(例えば、上方又は下方)からサンプルウエルを調べることができる。更に、システムは、蛍光標識又はプローブによって識別可能な生物的に関連する広範囲の分子を検出するようになっており、それは、核酸にも又はあらゆる特定の増幅処理にも限定されないものである。
Claims (18)
- サーマルサイクラの複数のウエル内に配置されたサンプルを分析する蛍光検出装置であって、
サーマルサイクラに取付可能な支持構造体と、
前記支持構造体上に移動可能に取付け可能な検出モジュールと、を備え、
該検出モジュールは、複数の励起/検出対を含み、各励起/検出対は、該検出モジュール内に配置された励起光発生器と、該検出モジュール内に配置された放出光検出器とを備え、
前記支持構造体が前記サーマルサイクラに取り付けられ前記検出モジュールは前記支持構造体に取付けられた時、該検出モジュールは、前記複数のウエルの異なるウエルと光学連通した状態で位置決めされるように移動可能である、
ことを特徴とする装置。 - 各放出光検出器は、異なる範囲の波長を検出するように構成されている、
請求項1に記載の蛍光検出装置。 - 各励起光発生器は、異なる範囲の波長で光を発生するように構成されている、
請求項1に記載の蛍光検出装置。 - 前記複数の励起/検出対は、各励起/検出対が前記複数ウエルの異なるものと光学接触した状態で同時に位置決め可能なように配置されている、
請求項1に記載の蛍光検出装置。 - 前記複数の励起/検出対は、該複数の励起/検出対の第1の対が前記複数のウエルのいずれか1つと光学接触した状態で位置決めされた時に、該励起/検出対の別の対が該複数のウエルのいずれとも光学接触しないように配置されている、
請求項1に記載の蛍光検出装置。 - 前記検出モジュールは、前記支持構造体に取外し可能に取付けられ、それによってユーザが該検出モジュールを異なる検出モジュールと交換することを可能にする、
請求項1に記載の蛍光検出装置。 - 較正要素であって、前記検出モジュールが前記較正要素と光学連通した状態で位置決めされるように移動可能であるように配置され、
既知の蛍光応答を供給する較正要素を備えている、
請求項1に記載の蛍光検出装置。 - 前記較正要素は、前記複数のウエルの2または3以上の間に位置している、
請求項7に記載の蛍光検出装置。 - 前記ウエルに対する前記検出モジュールの位置決めは、外部コンピュータにより制御される、
請求項1に記載の蛍光検出装置。 - 前記較正要素は、前記複数のウエルに対して周辺区域に位置している、
請求項7に記載の蛍光検出装置。 - 前記励起光発生器及び前記放出光検出器の作動は、外部コンピュータにより制御される、
請求項1に記載の蛍光検出装置。 - 溶液中のターゲット分子の存在を検出する方法であって、
ターゲット分子と結合するようになった蛍光プローブを各々含む複数のサンプルを準備するステップと、
検出モジュールが移動可能に取付けられたサーマルサイクラ装置の複数のサンプルウエルのそれぞれのウエルに各サンプルを配置するステップと、を備え、
前記検出モジュールは、複数の励起/検出対を含み、各励起/検出対は、該検出モジュール内に配置された励起光発生器と該検出モジュール内に配置された放出光検出器とを備え、
前記サーマルサイクラ装置を使用して反応を誘導するステップと、
前記検出モジュールを移動して前記複数の励起/検出対の少なくとも一つを作動させることにより、蛍光応答を検出するために前記複数のサンプルウエルを走査するステップと、を更に備え、
前記走査ステップ中に、前記検出モジュールが、前記励起/検出対の一つ以上が前記複数のサンプルウエルの各々と光学連通した状態に順次、位置決めされるように移動させられる、
ことを特徴とする方法。 - 前記ターゲット分子は、核酸配列である、
請求項12に記載の方法。 - 前記反応は、ポリメラーゼ連鎖反応(PCR)である、
請求項13に記載の方法。 - 前記走査するステップは、
第1の励起/検出対が前記サンプルウエルの1つと光学連通するように前記検出モジュールを位置決めするステップと、
前記第1の励起/検出対の前記励起光発生器を一時的に作動させるステップと、
前記第1の励起/検出対の前記放出光検出器を使用して蛍光応答を検出するステップと、
前記第1の励起/検出対が前記サンプルウエルの異なるサンプルウエルと光学連通するように前記検出モジュールを再位置決めするステップと、を含む、
請求項12に記載の方法。 - 前記検出モジュールが、前記第1の励起/検出対が1の前記サンプルウエルと光学連通するように位置決めされた時、第2の励起/検出対が、別のサンプルウエルと光学連通している、
請求項15に記載の方法。 - 前記検出モジュールが、前記第1の励起/検出対が1の前記サンプルウエルと光学連通するように位置決めされた時、第2の励起/検出対が、いずれのサンプルウエルとも光学連通していない、
請求項15に記載の方法。 - 前記走査ステップ中、前記検出モジュールは、前記第1の励起/検出対の前記励起光発生器が一時的に作動している間動作を続ける、
請求項15に記載の方法。
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US10724084B2 (en) | 2020-07-28 |
US20190136297A1 (en) | 2019-05-09 |
US20110160073A1 (en) | 2011-06-30 |
US20070059754A1 (en) | 2007-03-15 |
US8236504B2 (en) | 2012-08-07 |
US7148043B2 (en) | 2006-12-12 |
US8835118B2 (en) | 2014-09-16 |
CN1820082A (zh) | 2006-08-16 |
EP1620572A4 (en) | 2006-05-24 |
DE04751790T1 (de) | 2006-06-22 |
AU2004242098B2 (en) | 2008-09-25 |
US20180171387A1 (en) | 2018-06-21 |
WO2004104547A3 (en) | 2005-03-24 |
CA2525332A1 (en) | 2004-12-02 |
CN101241075A (zh) | 2008-08-13 |
DE602004004891T2 (de) | 2007-10-31 |
EP1620572A2 (en) | 2006-02-01 |
US20130143308A1 (en) | 2013-06-06 |
JP2007504477A (ja) | 2007-03-01 |
US10669576B2 (en) | 2020-06-02 |
CA2525332C (en) | 2014-01-07 |
US7749736B2 (en) | 2010-07-06 |
US20040224317A1 (en) | 2004-11-11 |
WO2004104547A2 (en) | 2004-12-02 |
CN101241075B (zh) | 2011-06-08 |
US20150152475A1 (en) | 2015-06-04 |
AU2004242098A1 (en) | 2004-12-02 |
EP1620572B1 (en) | 2007-02-21 |
DE602004004891D1 (de) | 2007-04-05 |
CN100378227C (zh) | 2008-04-02 |
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