JP2016540519A - アッセイ認識のための多重pcr反応のコード化法 - Google Patents
アッセイ認識のための多重pcr反応のコード化法 Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
Description
本明細書において用語「試料」は、核酸を含む検体又は培養物(例えば、微生物培養物)を含む。用語「試料」はまた、生物学的試料と環境試料の両方を含むことを意味する。試料は、合成起源の検体を含むことができる。生物学的試料は、全血、血清、血漿、臍帯血、絨毛膜絨毛、羊水、脳脊髄液、髄液、洗浄液(例えば、気管支洗浄液、胃洗浄液、腹膜洗浄液、管洗浄液、耳洗浄液、関節鏡洗浄液)、生検試料、尿、便、痰、唾液、鼻粘液、前立腺液、精液、リンパ液、胆汁、涙、汗、母乳、乳房流体、胚細胞、及び胎児細胞を含む。好適な実施態様において、生物学的試料は、血液、より好ましくは血漿である。本明細書において用語「血液」は、通常定義されるように、全血又は血液の任意の画分、例えば血清及び血漿などを包含する。血漿は、抗凝固剤で処理された血液の遠心分離に由来する全血の画分を指す。血清は、血液試料が凝固した後に残っている液体の水部分を指す。環境試料は、例えば表面物質、土壌、水及び産業試料、ならびに食品及び乳製品加工機器、装置、設備、器具、使い捨て及び非使い捨ての物品から得られる試料などの環境材料を含む。
PCR分析及び検出装置(例えば、Roche DiagnosticsのLightCycler(登録商標)装置)又は蛍光リーダーが、異なる濃度比を区別し同定することを可能にするであろう蛍光比を、2つの色素(JA270とCy5.5)の異なる濃度比が与えることができるかどうかを調べるために、実験が行われた。100μlの水中で0pmol〜20pmolの範囲の量を用いて、JA270とCy5.5との12の異なる濃度比が使用された。試験された異なる濃度比は表1に示される。
Claims (12)
- 複数の標的核酸のポリメラーゼ連鎖反応(PCR)増幅を行うための複数の反応混合物を調製する方法であって、
第1の標的核酸のPCR増幅を行うために必要な少なくとも1つの物質を含み、第1の蛍光色素と第2の蛍光色素とをさらに含む第1のマスターミックスであって、前記第1の色素と前記第2の色素は、前記第1の試薬溶液について特有である所定の濃度比で存在する、前記第1のマスターミックス溶液を提供する工程と、
第2の標的核酸のPCR増幅を行うために必要な少なくとも1つの物質を含み、前記第1の蛍光色素と前記第2の蛍光色素とをさらに含む第2のマスターミックスであって、前記第1の色素と前記第2の色素は、前記第1のマスターミックス溶液についての濃度比とは異なり、前記第2のマスターミックス溶液について特有である所定の濃度比で存在する、前記第2のマスターミックス溶液を提供する工程と、
前記第1のマスターミックス溶液を第1の反応混合物に加えて、前記第1の標的核酸のPCR増幅を行う工程と、
前記第2のマスターミックス溶液を第2の反応混合物に加えて、前記第2の標的核酸のPCR増幅を行う工程と
を含む、前記方法。 - 前記第1のマスターミックス溶液及び前記第2のマスターミックス溶液のいずれかにおいて、前記所定の濃度比が、ゼロ量の前記第1の蛍光色素を有し得る、請求項1に記載の方法。
- 前記第1のマスターミックス溶液及び前記第2のマスターミックス溶液のいずれかにおいて、前記所定の濃度比が、ゼロ量の前記第2の蛍光色素を有し得る、請求項1に記載の方法。
- 前記第1のマスターミックス溶液及び前記第2のマスターミックス溶液のいずれかにおいて、前記所定の濃度比が0〜5である、請求項1〜3のいずれか1項に記載の方法。
- 前記第1の蛍光色素がJA270であり、前記第2の蛍光色素がCys5.5である、請求項1〜4のいずれか1項に記載の方法。
- 標的核酸のポリメラーゼ連鎖反応(PCR)増幅のために使用されるマスターミックス溶液の同一性をコード化する方法であって、
所定の濃度比で存在する第1の蛍光色素と前記第2の蛍光色素とを、前記マスターミックス溶液中で一緒に混合する工程であって、前記所定の濃度比は、前記第2の蛍光色素を超える前記第1の蛍光色素の蛍光比を生成し、前記蛍光比は他のマスターミックス溶液から生成される蛍光比と区別できる、前記工程と、
前記第1の蛍光色素からの蛍光及び前記第2の蛍光色素からの蛍光を検出する工程と、
前記蛍光比を決定し、それにより前記マスターミックス溶液を同定する工程と
を含む、前記方法。 - 前記マスターミックス溶液がゼロ量の前記第1の蛍光色素を有する、請求項6に記載の方法。
- 前記マスターミックス溶液がゼロ量の前記第2の蛍光色素を有する、請求項6に記載の方法。
- 前記所定の濃度比が0〜5である、請求項6〜8のいずれか1項に記載の方法。
- 前記第1の蛍光色素がJA270であり、前記第2の蛍光色素がCys5.5である、請求項6〜9のいずれか1項に記載の方法。
- 複数のマスターミックス溶液を含む、複数の標的核酸のポリメラーゼ連鎖反応(PCR)増幅を行うためのキットであって、前記複数のマスターミックス溶液からの各1つのマスターミックス溶液は、特異的標的核酸のPCR増幅を行うために必要な少なくとも1つの物質を含み、第1の蛍光色素と第2の蛍光色素とをさらに含み、前記各1つのマスターミックス溶液について、前記第1の蛍光色素と前記第2の蛍光色素は、前記複数のマスターミックス溶液からの他のすべてのマスターミックス溶液とは異なる濃度比で存在し、かつ区別できる蛍光比を生成する、前記キット。
- 前記第1の蛍光色素がJA270であり、前記第2の蛍光色素がCys5.5である、請求項11に記載のキット。
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