双重实时荧光PCR对流感病毒检测及分型方法的研究  

Detection and subtype differentiation of influenza A virus by duplex real-time PCR

作　　者：沃恩康[1] 王怡婷[1] 吴海波[1] 尤金彪[1] 王巧刚[1] 郭潮潭[1] 

机构地区：[1]浙江省医学科学院生物工程研究所,杭州310013

出　　处：《中国卫生检验杂志》2013年第1期1-4,8,共5页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金项目(30872163;81071849;81272551);卫生部科学研究基金项目(WKJ2009-2-016);浙江省科技厅项目(2011F20012);浙江省公益性技术应用研究计划项目(2010C33166);浙江省卫生高层次创新人才培养工程项目;浙江省医学科学院青年基金

摘　　要：目的:建立双重实时荧光PCR方法用于流感病毒检测,实现对新甲型H1N1流感病毒,H1和H3亚型季节性流感病毒,H5和H9亚型禽流感病毒的亚型区分。方法:根据GenBank公布的基因序列,针对M基因保守区设计引物和探针,用于流感病毒检测和定量;针对HA基因设计引物和探针,用于病毒分型。建立和优化双重实时荧光PCR反应体系,进行敏感度和特异性评价,同时对32份已知亚型的流感病毒样本进行检测,验证所建方法的实用性。结果:基于M基因的实时荧光PCR方法定量范围为5×101到5×108个目的拷贝。经优化的双重实时荧光PCR方法对不同亚型流感病毒的检测灵敏度在50～500拷贝之间,各亚型之间没有交叉反应。建立的方法能够检测出本研究涉及的所有流感病毒标本,分型准确率达到96.9%。结论:建立的双重实时荧光PCR方法具有敏感度高,特异性好等优点,实现对流感病毒样本准确分型,适用于流感的鉴别诊断及病毒载量测定。Objective：To establish a duplex real-time PCR method for detection of influenza virus and differentiation of novel H1N1 influenza virus,human seasonal influenza viruses（subtypes H1 and H3） and avian influenza viruses（subtypes H5 and H9）.Methods： Based on the sequences alignment,the M gene-specific primer and probe were designed for the detection of all influenza viruses and virus titer quantitation.The HA gene-specific primers and probes were designed for subtype differentiation.The reaction conditions were optimized and the sensitivity,specificity and practicability of the assay were evaluated.Finally,the virus specimens were detected by the duplex real-time PCR method.Results： According to the M gene specific real-time PCR assay,the range of virus quantitation covered 5×101 to 5×108 copies.The established duplex real-time PCR assay was able to detect all influenza A viruses in the test.The detection limit was shown to be between 50 and 500 copies per reaction.According to the specificity assay results,there was no crossing reaction among different subtypes of influenza viruses.With this method,the subtype of virus specimens were differentiated correctly.Conclusion： The established duplex real-time PCR method had advantages in sensitivity and specificity.It was suitable for influenza virus differential diagnosis and determination of viral load.

关 键 词：流感病毒 双重实时荧光PCR 分型 

分 类 号：R446.5[医药卫生—诊断学]