A (H3N2)亚型流感病毒R292K和N294S突变位点TaqMan-MGB探针检测方法的建立.pdfVIP

昆明医科大学学报 2016 ,37( 10) :27- 30 CN 53 -1221/R Journal of Kunming Medical University A (H3N2)亚型流感病毒 R292K 和 N294S 突变位点TaqMan-MGB 探 针检测方法的建立 赵晓南，宁德明，李多，罗春藤，李娟，伏晓庆，徐闻，王瑾 (云南省疾病预防控制中心，云南昆明 650022) [摘要]目的建立A 例如2) 亚型流感病毒 R292K 和 N294S 突变位点 TaqMan-MGB 探针检测方法.方法 自 NCBI 获取的6 株流感疫苗株及 2012 年- 2015 年本实验室分离的 17 株，共计23 条A 例如2) 亚型毒株NA 基因全序列.根据序列比对结果，特异性设计针对R292K 和 N294S 突变位点的 TaqMan-MGB 探针及引物进行3 重realtime RT-PCR 检测方法.使用倍比稀释的方法检测其敏感性.并利用临床标本，其他亚型流感及其他常见呼 吸道病毒验证该方法的特异性.结果设计的TaqMan-MGB 探针可以检测到 1σ (1∞o copies) 稀释度的对应模 板，使用其他亚型流感病毒与常见呼吸道病毒验证该引物无交叉反应.通过对57 份日常监测分离毒株进行检测， 未发现耐药位点突变株.结论建立了A (H3N2) 亚型流感病毒 R292K 和 N294S 突变位点TaqMan-MGB 探针检 测方法;验证了该引物探针具有较高的敏感性与特异性，具有实际应用潜力. [关键词] H3N2; 突变;实时荧光RT-PCR; [中图分类号] R51L7 [文献标志码] A [文章编号] 2095-6 1OX (2016) 10一∞27-04 Establishment of Detection Method for R292K and N294S Mutation Sites in H3N2 I nf1uenza A Virus with TaqMan-MGB Probes ZHAO Xiao-nan , NING De-mi吨， LIDuo， LUOChun-rui , LIJuan , FUXiao-qing , XUWen , WANGJin (Yunnan Centersfor Diseωe Control and 丹evention ， Kunming Yunnan 650022 , Chinα) [AbstractJ Objective To establish a detection method to R292K and N294S mutation sites in H3N2 influenza A virus with TaqMan-MGB probes. Method NA gene sequences of 23 A (H3N2) influenza strains were acquired from NCBI database and lab isolations. According to sequence alignment results , specific TaqMan-MGB probes and primers were designed for R292K and N294S mutation sites in multiple realtime PCR method. The sensitivity was detected using the ten times dilution method. Clinical specimens , other subtypes of influenza and other common respiratory viruses were used to verify the spec