JP4299003B2 - ORF6LeftPCR(配列番号12)またはORF6RightPCR(配列番号13)を用いた肺炎マイコプラズマの増幅および検出 - Google Patents
ORF6LeftPCR(配列番号12)またはORF6RightPCR(配列番号13)を用いた肺炎マイコプラズマの増幅および検出 Download PDFInfo
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- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Description
配列番号1〜2は、ORF6遺伝子内配列の増幅用に上流プライマーとして使用するオリゴヌクレオチド配列である。配列番号3〜4は、ORF6遺伝子内配列の増幅用に下流プライマーとして使用するオリゴヌクレオチド配列である。配列番号5〜6は、SDA増幅用に上流バンパープライマーとして使用するオリゴヌクレオチド配列である。配列番号7〜8は、SDA増幅用に下流バンパープライマーとして使用するオリゴヌクレオチド配列である。配列番号9〜10は、ORF6遺伝子内配列の増幅および検出用のシグナルプライマー配列である。配列番号11は、前記シグナルプライマーのいずれかと共に使用した場合、ORF6遺伝子内配列の検出用に設計されたレポータープローブ用の配列となる。配列番号12は、ORF6遺伝子内配列のPCR増幅用に上流プライマーとして使用するオリゴヌクレオチド配列である。配列番号13は、ORF6遺伝子内配列のPCR増幅用に下流プライマーとして使用するオリゴヌクレオチド配列である。
以下の実施例は、ここで記載した本発明の特定の実施形態を例示するものである。当分野の技術者には言うまでもないことであるが、様々な変更および修正が可能であり、記載した本発明の範囲内であると意図される。
ORF6遺伝子の肺炎マイコプラズマに対する特異性を判定するために、配列番号12および配列番号13のプライマーを用いて、数種の肺炎マイコプラズマ株から得たDNAのPCR増幅を行った。簡潔に述べれば、PCR条件は以下のとおり、配列番号12および配列番号13のプライマー各10μM、dATP、dTTP、dCTP、およびdGTP各200nM、AmpliTAQ DNAポリメラーゼ(Perkin Elmer)5単位、1:10希釈のATCCの保存生物(organism stock)1μlを、1×AmpliTAQ緩衝液を含む50μlの反応体積とした。PCRは、MJ Research PTC−200 Peltier Thermal Cyclerにおいて、95℃で5分間の1サイクルに続いて95℃で45秒、52℃で45秒、72℃で60秒の35サイクルからなる温度循環条件で行った。各PCR産物9μlを1μlの添加色素と混合し、アガロースゲル電気泳動法によって分析した。100bpラダー(GibcoBRL)ゲルマーカーを使用して、産物の大きさを推定した。98ボルトで約45分間、ゲル上で泳動を行った。図2は、8種の肺炎マイコプラズマATCC株のPCR増幅から得た産物の1%アガロースゲル分析の結果を示している。表1は、試験した株と、図2の標識されたバンドに対応するバンドの同定を示す。
実施例1で得られたPCR産物の配列決定を行い、MegAlignソフトウェアプログラム(DNAStar)を使用して、8種のORF6配列の整列をコンパイルした。
ORF6ターゲット配列用の前述した2つのSDA系を含むオリゴヌクレオチドの特異性を評価するために、当技術分野でよく知られている(非特許文献11、12、13、14)Basic Local Alignment Search Tool(「BLAST」)を使用して、核酸配列の相同性を評価した。BLASTNを使用して、ヌクレオチド照会配列をヌクレオチド配列データベースと対照した。BLASTNプログラムは、照会核酸配列と、好ましくは核酸配列データベースから得た試験配列とで類似する区域、すなわちここでは「高得点区域対」と呼ぶものを同定することによって相同配列を特定する。その多くが当技術分野で知られている得点行列によって高得点区域対を同定(すなわち整列)することが好ましい。使用する得点行列(scoring matrix)は、Blosum62行列(非特許文献15)であることが好ましい。BLASTNプログラムは、同定された高得点区域対すべての統計的有意性を評価し、好ましくは、相同性百分率など、使用者が指定した有意性の閾値を満たす区域を選択する。高スコア区域対の統計的有意性は、Karlin(非特許文献16)の統計的有意性の式を使用して評価することが好ましい。
Claims (8)
- ORF6LeftPCR(配列番号12)またはORF6RightPCR(配列番号13)のターゲット結合配列からなることを特徴とするオリゴヌクレオチド。
- ORF6LeftPCR(配列番号12)またはORF6RightPCR(配列番号13)のターゲット結合配列と、制限エンドヌクレアーゼによってニッキング可能な制限エンドヌクレアーゼ認識部位とからなることを特徴とする請求項1に記載のオリゴヌクレオチド。
- a)ORF6LeftPCR(配列番号12)のターゲット結合配列を含む第1のプライマーと、
b)ORF6RightPCR(配列番号13)のターゲット結合配列を含む第2のプライマーと、を含むことを特徴とする増幅プライマーの対。 - a)ORF6LeftPCR(配列番号12)のターゲット結合配列を含む第1のプライマーおよび制限エンドヌクレアーゼによってニッキング可能な制限エンドヌクレアーゼ認識部位と、
b)ORF6RightPCR(配列番号13)のターゲット結合配列を含む第2のプライマーおよび制限エンドヌクレアーゼによってニッキング可能な制限エンドヌクレアーゼ認識部位と、を含むことを特徴とする増幅プライマーの対。 - a)ORF6LeftPCR(配列番号12)のプライマー、
b)ORF6RightPCR(配列番号13)のプライマー、
c)ORF6ADPT1(配列番号9)、配列番号9に相補的な核酸、ORF6ADPT2(配列番号10)、および配列番号10に相補的な核酸からなる群から選択された1または2以上のシグナルプライマー、
を含むことを特徴とするキット。 - e)レジオネラ菌に特異的な核酸配列の増幅に特異的なプライマーの対、
f)百日咳菌に特異的な核酸配列の増幅に特異的なプライマーの対、
g)クラミジア感染の指標となる核酸配列の増幅に特異的なプライマーの対
をさらに含むことを特徴とする請求項5に記載のキット。 - 肺炎マイコプラズマのターゲット核酸配列を増幅する方法であって、
a)核酸に、
i)ORF6LeftPCR(配列番号12)のターゲット結合配列からなる第1の増幅プライマーと、
ii)ORF6RightPCR(配列番号13)のターゲット結合配列からなる第2の増幅プライマーと、をハイブリダイズさせること、ならびに
b)ハイブリダイズされた第1および第2の増幅プライマーをターゲット核酸配列上で伸長させ、それによってターゲット核酸配列を増幅すること、を含むことを特徴とする方法。 - 肺炎マイコプラズマのターゲット核酸配列を増幅する方法であって、
a)核酸に、
i)ORF6LeftPCR(配列番号12)のターゲット結合配列からなる第1の増幅プライマーおよび制限エンドヌクレアーゼによってニッキングされる制限エンドヌクレアーゼに対する認識部位と、
ii)ORF6RightPCR(配列番号13)のターゲット結合配列からなる第2の増幅プライマーおよび制限エンドヌクレアーゼによってニッキングされる制限エンドヌクレアーゼに対する認識部位と、をハイブリダイズさせること、ならびに
b)ハイブリダイズされた第1および第2の増幅プライマーをターゲット核酸配列上で伸長させ、それによってターゲット核酸配列を増幅すること、を含むことを特徴とする方法。
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US28360101P | 2001-04-13 | 2001-04-13 | |
PCT/US2002/011630 WO2002086441A2 (en) | 2001-04-13 | 2002-04-11 | Amplification and detection of mycoplasma pneumoniae |
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JP2008231464A Pending JP2009060901A (ja) | 2001-04-13 | 2008-09-09 | 肺炎マイコプラズマの増幅および検出 |
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US (1) | US20040219544A1 (ja) |
EP (1) | EP1390710B1 (ja) |
JP (2) | JP4299003B2 (ja) |
AT (1) | ATE319731T1 (ja) |
AU (1) | AU2002303339B2 (ja) |
CA (1) | CA2443776A1 (ja) |
DE (1) | DE60209711T2 (ja) |
DK (1) | DK1390710T3 (ja) |
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CN104946769B (zh) * | 2015-06-30 | 2017-11-07 | 中国疾病预防控制中心传染病预防控制所 | 肺炎支原体快速检测和基因分型的试剂盒 |
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AU702896B2 (en) * | 1995-09-21 | 1999-03-11 | Becton Dickinson & Company | Detection of nucleic acids in cells by thermophilic strand displacement amplification |
US5817463A (en) * | 1996-06-28 | 1998-10-06 | Abbott Laboratories | Nucleic acid primers and probes for detecting Mycoplasma pneumoniae |
FR2775002B1 (fr) * | 1998-02-19 | 2003-01-10 | France Etat | Procede de diagnostic d'agents pathogenes respiratoires par biologie moleculaire |
US6277582B1 (en) * | 2000-07-27 | 2001-08-21 | Becton, Dickinson And Company | Amplification and detection of mycoplasma pneumoniae targeting the P1 gene |
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NO20034555D0 (no) | 2003-10-10 |
CA2443776A1 (en) | 2002-10-31 |
DE60209711T2 (de) | 2006-11-16 |
DK1390710T3 (da) | 2006-07-17 |
EP1390710B1 (en) | 2006-03-08 |
AU2002303339B2 (en) | 2008-08-07 |
ATE319731T1 (de) | 2006-03-15 |
EP1390710A4 (en) | 2005-07-13 |
WO2002086441A2 (en) | 2002-10-31 |
JP2009060901A (ja) | 2009-03-26 |
ES2257549T3 (es) | 2006-08-01 |
WO2002086441A3 (en) | 2003-04-17 |
NO330237B1 (no) | 2011-03-14 |
JP2005508136A (ja) | 2005-03-31 |
DE60209711D1 (de) | 2006-05-04 |
US20040219544A1 (en) | 2004-11-04 |
NO20034555L (no) | 2003-12-09 |
EP1390710A2 (en) | 2004-02-25 |
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