逆转录环介导等温核酸扩增技术(RT-LAMP)在H5N1禽流感病毒基因检测中的应用Evaluation of Reverse Transcription Loop-mediated Isothermal Amplification for Detection of Avian Influenza A H5N1 Virus
李启明,马学军,高寒春,周蕊,匡治州,侯云德
摘要(Abstract)：
建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测。该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应。对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green I为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试。结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性。此方法的灵敏度可达到能检测10个拷贝RNA分子水平。因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法。
关键词(KeyWords)： H5N1亚型禽流感病毒;逆转录环介导等温核酸扩增技术;实时监控
基金项目(Foundation): 科技部禽流感防治重大专项(2004BA519A47,2004BA519A34)
作者(Author): 李启明,马学军,高寒春,周蕊,匡治州,侯云德
参考文献(References)：
- [1]Notomi T,Okayama H,Masubuchi H,et al.Loop-me-diated isothermal amplification of DNA[J].Nucl Acids Res,2000,28:E63.
- [2]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplification using loop pri mers[J].Mol Cell Probes,2002,16:223-229.
- [3]Nagamine K,Watanabe K,Ohtsuka K,et al.Loop-me-diated isothermal amplification reaction using a nondena-tured template[J].Clin Chem,2001,47:1742-1743.
- [4]Mori Y,Kitao M,Tomita N,et al.Real-ti me turbidi m-etry of LAMP reaction for quantifying template DNA[J].J Biochem Bioph Meth,2004,59:145-157.
- [5]Viseshakul N,Thanawongnuwech R,Amonsin A,et al.The genome sequence analysis of H5N1avianinfluen-za Avirus isolated fromthe outbreak among poultry pop-ulations in Thailand[J].Virology,2004,328:169-176.
- [6]Saw T A,Li m W,Shortridge K,et al.Isolation of avi-an influenza A(H5N1)viruses from humans-Hong Kong,May-December1997[J].MMWR Morb Mortal Wkly Rep,1997,46:1204-1207.
- [7]Tran T H,Nguyen T,Nguyen TD,et al.Avian influ-enza A(H5N1)in10patients in Vietnam[J].N Engl J Med,2004,350:1179-1788.
- [8]Chotpitayasunondh T,Lochindarat S,Srisan P,et al.Cases of influenza A(H5N1)-Thailand,2004[J].MM-WR Morb Mortal Wkly Rep,2004,53:100-103.
- [9]Munch M,Nielsen L P,Handberg KJ,et al.Detection and subtyping(H5and H7)of avian type Ainfluenza vi-rus by reverse transcription-PCR and PCR ELISA[J].Arch Virol,2001,146:87-97.
- [10]Kwok S,Higuchi R.Avoiding false positives with PCR[J].Nature,1989,339:237-238.
- [11]Ward C L,Dempsey M H,Ring CJ,et al.Design and performance testing of quantitative real ti me PCR as-says for influenza Aand B viral load measurement[J].J Clin Virol,2004,29:179-188.
- [12]Cooper L A,Subbarao K.A si mple restriction frag-ment length polymorphism-based strategy that can dis-tinguish the internal genes of human H1N1,H3N2,and H5N1influenza A viruses[J].J Clin Microbiol,2000,38:2579-2583.
- [13]Spackman E,Senne D A,Myers T J,et al.Develop-ment of a real-ti me reverse transcriptase PCRassay for type Ainfluenza virus and the avian H5and H7he-magglutinin subtypes[J].J Clin Microbiol,2002,40:3256-3260.