实时RT-PCR检测甲型H3N2流感病毒R292K耐药位点  

Development and evaluation of a novel real-time reverse-transcriptase PCR assay for detection of R292K mutation of the H3N2 influenza A virus

作　　者：刘娟[1,2] 姜涛[1] 徐莉娟[1] 于曼[1] 秦鄂德[1] 秦成峰[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [2]空军总医院输血科,北京100142

出　　处：《军事医学》2011年第6期432-436,共5页Military Medical Sciences

基　　金：“重大新药创制”科技重大专项资助项目(2009ZX10004-204,2008ZX10004-001);国家自然科学基金资助项目(81000722)

摘　　要：目的建立快速检测甲型H3N2流感病毒神经氨酸酶(NA)奥司他韦耐药突变位点的实时RT-PCR方法。方法通过设计特异性靶向NAR292K突变位点的Taq-MGB探针进行实时RT-PCR反应,并利用模拟样本和临床标本进行灵敏性和特异性评价。结果与结论建立了快速检测NAR292K位点的实时RT-PCR检测方法;本方法灵敏性高,可检测低至500拷贝/μl;特异性好,与无耐药突变的甲型H3N2流感病毒及其他呼吸道病毒均无交叉反应,为流感耐药监测提供了有效工具。ObjectiveTo develop a rapid and sensitive assay for detection of R292K oseltamivir-resisitant mutation of the H3N2 influenza A virus.MethodUsing specific Taq-MGB probe targeting R292K mutation,a real-time RT-PCR assay was developed.The sensitivity of the assay was analyzed using a 10-fold dilution series of plasmid DNA containing partial neuramidinase（NA） sequence with R292K mutation.The specificity of the assay was also analyzed with H3N2 influenza virus without R292K mutation and other common respiratory viruses.Results and ConclusionThe detection limit of the R292K rRT-PCR assay was 500 copies/μl.No cross-reaction with H3N2 virus without R292K mutation and other five respiratory viruses was observed.The R292 real-time RT-PCR assay is specific and sensitive,and potentially useful in influenza resistance surveillance and clinical diagnosis.

关 键 词：流感病毒 H3N2亚型 抗病毒药物 耐药性 实时RT-PCR 

分 类 号：R511.7[医药卫生—内科学]