JP5859424B2 - 毒素産生クロストリジウムディフィシル菌の検出及び特性解析方法 - Google Patents
毒素産生クロストリジウムディフィシル菌の検出及び特性解析方法 Download PDFInfo
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Description
Claims (10)
- サンプル中の毒素産生クロストリジウムディフィシル菌を検出し、特性解析する方法であって、
a.前記サンプルが準備されるステップと、
b.多重PCRアッセイにおいて、
i.前記サンプルが、細胞毒素tcdB遺伝子の有無について解析され、
ii.前記サンプルが、tcdC遺伝子の、
a)SEQ ID No. 1のヌクレオチド330からヌクレオチド347までの18bp欠失、
b)SEQ ID No. 1のヌクレオチド301からヌクレオチド336までの36bp欠失、
c)SEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失、
d)SEQ ID No. 1のヌクレオチド313からヌクレオチド366までの54bp欠失、
e)SEQ ID No. 1の位置117における単一ヌクレオチド欠失、
の1又は複数の有無について解析される、ステップと、
を含み、前記ステップbにおける多重PCR増幅が定量リアルタイムPCRであり、
前記サンプルが、エンテロトキシンtcdA遺伝子の1.8kb欠失の有無について付加的に解析される、方法。 - 前記サンプルが、バイナリトキシンcdtA及びcdtBの有無について付加的に解析される、請求項1に記載の方法。
- 前記サンプルが、
a.SEQ ID No. 1のヌクレオチド330からヌクレオチド347までの18bp欠失の有無、
b. SEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失の有無、
c.SEQ ID No. 1の位置117の単一ヌクレオチド欠失の有無、
d.細胞毒素tcdB遺伝子の有無、
e.1.8kb tcdA欠失の有無、及び
f.cdtA/Bバイナリトキシン遺伝子の有無、
について解析される、請求項1乃至2のいずれか1項に記載の方法。 - a.前記tcdB遺伝子の配列が存在し、tcdA欠失が無く、SEQ ID No. 1の位置117の単一ヌクレオチド欠失が無く、18bp欠失が無く、39bp欠失が無い場合、前記サンプルは、毒素産生クロストリジウムディフィシルとしてスコアリングされること、
b.前記tcdB遺伝子の配列が存在し、tcdA欠失が無く、SEQ ID No. 1の位置117の単一ヌクレオチド欠失が存在し、18bp欠失が存在し、cdtA/Bバイナリトキシン遺伝子が存在する場合、前記サンプルは、リボタイプ027クロストリジウムディフィシル菌としてスコアリングされること、
c.前記tcdB遺伝子の配列が存在し、tcdA欠失が存在し、SEQ ID No. 1の位置117の単一ヌクレオチド欠失が無く、18bp欠失が無く、SEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失が無く、cdtA/Bバイナリトキシン遺伝子が無い場合、前記サンプルは、リボタイプ017クロストリジウムディフィシル菌としてスコアリングされること、及び
d.前記tcdB遺伝子の配列が存在し、tcdA欠失が無く、SEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失が存在し、cdtA/Bバイナリトキシン遺伝子が存在する場合、前記サンプルは、リボタイプ078クロストリジウムディフィシル菌としてスコアリングされること、
の前記a乃至前記dの個々のスコアリングが別個に実施される、請求項3に記載の方法。 - 前記多重PCRアッセイの多重増幅反応は、反応混合物中の蛍光指標の存在下で密閉システムにおいて行われ、前記蛍光指標は、前記増幅反応において各アンプリコンの存在及び/又は量に関連する光学的信号を生成し、前記増幅反応において前記蛍光指標の光学的信号を監視することが可能である、請求項1乃至4のいずれか1項に記載の方法。
- 前記密閉システムは、ユーザに、前記スコアリングの付与を示す光学的出力を与える、請求項4を引用する請求項5に記載の方法。
- 前記多重PCRアッセイの増幅産物が、60乃至200bpの大きさである、請求項1乃至6のいずれか1項に記載の方法。
- 前記サンプルは、人間又は動物の排泄物である、請求項1乃至7のいずれか1項に記載の方法。
- (i)細胞毒素tcdB遺伝子、
(ii)tcdA遺伝子の1.8kb欠失、
(iii)tcdC遺伝子のSEQ ID No. 1のヌクレオチド330からヌクレオチド347までの18bp欠失、
(iv)tcdC遺伝子のSEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失、及び
(v)SEQ ID No. 1の位置117の単一ヌクレオチド欠失、を増幅し及び/又は検出するためのプライマ及び/又はプローブと、
(vi)バイナリトキシンcdtA/B遺伝子を検出するためのプライマ及び/又はプローブと、
を含む1又は複数のチャネル又はチャンバを有する密閉システム増幅カートリッジ。 - 請求項3に記載の方法を実施するためのキットであって、
(i)細胞毒素tcdB遺伝子、
(ii)tcdA遺伝子の1.8kbの欠失、
(iii)tcdC遺伝子のSEQ ID No. 1のヌクレオチド330からヌクレオチド347までの18bp欠失、
(iv)tcdC遺伝子のSEQ ID No. 1のヌクレオチド341からヌクレオチド370までの39bp欠失、及び
(v)SEQ ID No. 1の位置117の単一ヌクレオチド欠失、を増幅し及び/又は検出するためのプライマ及び/又はプローブと、
(vi)バイナリトキシンcdtA/B遺伝子を検出するためのプライマ及び/又はプローブと、
を含むキット。
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PCT/IB2010/051396 WO2010116290A1 (en) | 2009-04-07 | 2010-03-31 | Method for the detection and characterization of a toxinogenic clostridium difficile strain |
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WO2012087135A1 (en) | 2010-12-22 | 2012-06-28 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Genetic markers specific for clostridium difficile ribotypes 027 (nap01/b1; rt 027) and 078 (nap7/8; rt 078) and their use |
GB201110712D0 (en) | 2011-06-23 | 2011-08-10 | Univ Ulster | Diagnostic methods |
EP4083230A1 (en) | 2011-07-06 | 2022-11-02 | Quest Diagnostics Investments Incorporated | Direct amplification and detection of viral and bacterial pathogens |
GB201207998D0 (en) * | 2012-05-08 | 2012-06-20 | Univ Cardiff | A screening method for the detection of clostridium difficle |
US9133527B2 (en) * | 2012-05-11 | 2015-09-15 | Techlab, Inc. | Cell wall protein CwpV (CD0514) as a diagnostic marker for Clostridium difficile ribotype 027 |
KR101955329B1 (ko) | 2012-06-08 | 2019-03-07 | 삼성전자주식회사 | 클로스트리디움 디피실리 균주 검출용 조성물과 키트 및 이를 이용한 검출 방법 |
CN102952886A (zh) * | 2012-11-28 | 2013-03-06 | 中华人民共和国张家港出入境检验检疫局 | 艰难梭菌肠毒素a、b双重荧光定量pcr检测方法及检测用试剂盒 |
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TWI504751B (zh) * | 2013-09-25 | 2015-10-21 | Univ Nat Cheng Kung | 檢測困難梭狀桿菌核醣分型027之序列、技術平台及其方法 |
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US20190211377A1 (en) * | 2016-12-22 | 2019-07-11 | Roche Molecular Systems, Inc. | Cobra probes to detect a marker for epidemic ribotypes of clostridium difficile |
US20200318171A1 (en) * | 2017-12-15 | 2020-10-08 | Gen-Probe Incorporated | Compositions and Methods for Detecting Toxigenic Clostridium Difficile |
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