JP2010537648A - クロストリジウム・ディフィシレの毒素原性菌株の検出 - Google Patents
クロストリジウム・ディフィシレの毒素原性菌株の検出 Download PDFInfo
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Abstract
Description
任意のPCR診断アッセイにおけるプライマーおよびプローブの設計は、常に感度と特異性との妥協であり、迅速性およびハイブリダイゼーション温度が考慮される必要がある。その蓄積を最大限にし、サイクル時間を低減するために、一般的には最も短いアンプリコンを設計する。プライマーと分子標識プローブ(下記で定義)との間の融解温度の温度差は、一般的に可能な限り大きい。これは、標識基部の長さおよびGC含量を変えることにより達成することができる。プライマーおよびプローブのそのような最適化には、核酸配列のデータベース解析および計算から得られる特定量の理論データが必要とされる。関連データの概要を下記に提供する。
tcdB配列および内部対照pDIFFaを標的とする分子標識を、配列データベースおよびソフトウェアOligo(商標)(バージョン6.0;National Biosciences社製)を使用して設計した。分子標識プローブを選択する際に考慮した様々な基準を下記に要約する。
アッセイの適切な設計には、増幅反応に関する潜在的な問題の検証も必要である。増幅効率は、プライマー、プローブ、およびそれぞれの標的間の二次構造および不一致によって大きく影響される場合がある。そのようなことが発生するのを防止するため、すべてのプライマーについて、ヘアピン構造を形成する能力を、IDT社のウェブサイトで利用可能なIDT社製OligoAnalyzer3.0ソフトウェアを用いて評価した。使用したパラメーターは、0.25μMの各プライマー、100mM Na+、5.5mM MgCl2、標的DNA、57℃のハイブリダイゼーション温度であった。ハイブリダイゼーションは、関与する分子の熱力学的特徴に依存するため、二次構造または望ましくない一致は、このようにして予測および回避することができる。加えて、溶液中で生じるPCRアッセイの全反応において、ギブス自由エネルギー(ΔGと記し、kcal/molで表す)は、一致が生じる可能性が高いかどうかを予測する。負のΔG値は、提唱構造または一致が形成することを示し、正のΔG値は、提唱構造が熱力学的に不安定であり、一致が生じない可能性が高いことを示す。プライマーKERLA−tcdB−2873は、2つのヘアピン構造を形成する可能性があり(ΔG=0.86および0.89kcal/mol)、プライマーKENP−tcdB−3012は、2つのヘアピン構造を形成する可能性がある(ΔG=1.9および2.35kcal/mol)。これらの構造は、すべて熱力学的に不安定である(正のΔG)。
22個の異なるC.ディフィシレ毒素遺伝子変異型を、表4に示されているプローブを用いて試験した。すべての毒素遺伝子変異型で陽性結果が得られたが、いかなる近縁種、C.ソルデリイ、C.ディフィシレA−/B−菌株、または非毒素原性C.ディフィシレ菌株でも陽性結果は得られなかった。したがって、プローブは、C.ディフィシレの毒素原性菌株に特異的である。
凍結乾燥された試薬を、225μl希釈液を用いて再構成して、リアルタイムPCRアッセイに使用された以下の緩衝液:116mMトリスHCl、pH8.3、11.6mM KCl、3.48mM MgCl2、5.8mM NH2SO4を調製し、その後、25μl等量に分割した。0.5、2.5、5、10、または20コピーのC.ディフィシレ鋳型DNAを、5つの反復反応液の各々に添加した。
Claims (23)
- C.ディフィシレ毒素B(TcdB)遺伝子にハイブリダイズすることが可能である長さが最大で100核酸塩基のオリゴヌクレオチドプローブまたはプライマーであって、配列番号1〜33からなる群から選択される配列、または配列番号1〜33からなる群から選択される配列と少なくとも85%の同一性を示す配列を含むオリゴヌクレオチドプローブまたはプライマー。
- 配列番号1〜33からなる群から選択される配列、または配列番号1〜33からなる群から選択される配列と少なくとも85%の同一性を示す配列を有する、請求項1に記載のオリゴヌクレオチドプローブまたはプライマー。
- 配列番号1〜33からなる群から選択される配列を有する、請求項1に記載のオリゴヌクレオチドプローブまたはプライマー。
- 生体試料中のC.ディフィシレの毒素原性菌株の存在を決定する方法であって、
前記試料を、C.ディフィシレ毒素B(TcdB)遺伝子に結合することが可能である少なくとも1対のプライマーと接触させるステップであって、前記少なくとも1対のプライマーの各プライマーが、長さが最大で100核酸塩基であり、C.ディフィシレ毒素B(TcdB)遺伝子に結合することが可能であり、前記少なくとも1対のプライマーの各プライマーが、配列番号1〜33からなる群から選択される配列、または配列番号1〜33からなる群から選択される配列と少なくとも85%の同一性を示す配列を含むステップと、
前記試料由来の標的核酸を増幅するステップと、
前記試料中のC.ディフィシレの前記毒素原性菌株の存在の指標として、増幅産物(複数可)の存在または量を検出するステップと
を含む方法。 - 前記試料が、糞便、痰、末梢血、血漿、血清、リンパ節、呼吸組織、および滲出液からなる群から選択される、請求項4に記載の方法。
- 前記試料が糞便試料である、請求項5に記載の方法。
- 前記試料を1対のプライマーと接触させる、請求項4に記載の方法。
- 前記増幅が、ポリメラーゼ連鎖反応(PCR)、リガーゼ連鎖反応(LCR)、鎖置換増幅(SDA)、レプリカーゼ媒介増幅、および転写媒介増幅からなる群から選択される方法によって実行される、請求項4に記載の方法。
- 前記増幅がPCRを使用して実行される、請求項8に記載の方法。
- 前記PCRが、AFLP、Alu−PCR、非対称PCR、コロニーPCR、DD−PCR、デジェネレートPCR、ホットスタートPCR、In situ PCR、インバースPCR、ロングPCR、マルチプレックスPCR、ネステッドPCR、PCR−ELISA、PCR−RFLP、PCR−一本鎖高次構造多型(PCR−SSCP)、定量的競合PCR(QC−PCR)、cDNA末端迅速増幅−PCR(RACE−PCR)、多型DNAランダム増幅−PCR(RAPD−PCR)、リアルタイムPCR、反復遺伝子外パリンドロームPCR(Rep−PCR)、逆転写PCR(RT−PCR)、TAIL−PCR、タッチダウンPCR、およびベクトレットPCRからなる群から選択される、請求項9に記載の方法。
- 前記PCRが定量的リアルタイムPCR(QRT−PCR)である、請求項10に記載の方法。
- 各プライマーが、増幅自体に対して著しい影響を与えずに増幅産物の増幅後操作を可能にする外来性ヌクレオチド配列を導入する、請求項7に記載の方法。
- 前記プライマー対が配列番号30および31を含む、請求項7に記載の方法。
- 前記プライマー対が配列番号32および33を含む、請求項7に記載の方法。
- 前記プライマー対の各プライマーに、5’末端に蛍光体、3’末端に蛍光消光体を含む相補的配列が隣接している、請求項14に記載の方法。
- 生体試料中のC.ディフィシレの毒素原性菌株の存在を核酸に基づく増幅によって検出する際の、C.ディフィシレ毒素B(TcdB)遺伝子にハイブリダイズすることが可能である少なくとも1対のプライマーの使用であって、前記少なくとも1対のプライマーの各プライマーは、長さが最大で100核酸塩基であり、前記少なくとも1対のプライマーの各プライマーが、配列番号1〜33からなる群から選択される配列、または配列番号1〜33からなる群から選択される配列と少なくとも85%の同一性を示す配列を含む使用。
- 前記試料が、糞便、痰、末梢血、血漿、血清、リンパ節、呼吸組織、および滲出液からなる群から選択される、請求項16に記載の使用。
- 前記試料が糞便試料である、請求項17に記載の使用。
- 1対の前記プライマーが使用される、請求項16に記載の使用。
- 前記核酸に基づく増幅が、ポリメラーゼ連鎖反応(PCR)、リガーゼ連鎖反応(LCR)、鎖置換増幅(SDA)、レプリカーゼ媒介増幅、および転写媒介増幅からなる群から選択される、請求項16に記載の使用。
- 前記核酸に基づく増幅がPCRである、請求項20に記載の使用。
- 前記PCRが、AFLP、Alu−PCR、非対称PCR、コロニーPCR、DD−PCR、デジェネレートPCR、ホットスタートPCR、In situ PCR、インバースPCR、ロングPCR、マルチプレックスPCR、ネステッドPCR、PCR−ELISA、PCR−RFLP、PCR−一本鎖高次構造多型(PCR−SSCP)、定量的競合PCR(QC−PCR)、cDNA末端迅速増幅−PCR(RACE−PCR)、多型DNAランダム増幅−PCR(RAPD−PCR)、リアルタイムPCR、反復遺伝子外パリンドロームPCR(Rep−PCR)、逆転写PCR(RT−PCR)、TAIL−PCR、タッチダウンPCR、およびベクトレットPCRからなる群から選択される、請求項21に記載の使用。
- 前記PCRが定量的リアルタイムPCR(QRT−PCR)である、請求項22に記載の使用。
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US20090208948A1 (en) | 2009-08-20 |
EP2195433B1 (en) | 2014-12-24 |
AU2008295396A2 (en) | 2010-05-13 |
CA2698232A1 (en) | 2009-03-12 |
CA2698232C (en) | 2018-06-12 |
US20150292004A1 (en) | 2015-10-15 |
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