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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
(云南省疾病预防控制中心,云南 昆明 650022)
摘要:
[摘要]目的 建立A(H3N2)亚型流感病毒R292K和N294S突变位点TaqMan-MGB探针检测方法.方法 由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(H3N2)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107(1000 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:  [关键词]H3N2  突变  实时荧光RT-PCR  
DOI:
分类号:
基金项目:[基金项目]云南省应用基础研究计划青年项目(2013FD096)
Establishment of Detection Method for R292K and N294S Mutation Sites in H3N2 Influenza A Virus with TaqMan-MGB Probes
ZHAO Xiao-nan
(Yunnan Centers for Disease Control and Prevention,Kunming Yunnan 650022,China)
Abstract:
[Abstract]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 specificity of the method.Results The designed probes could detect 107 (1000 copies) dilution of the corresponding template. Other subtypes of influenza virus and common respiratory viruses showed no cross reaction. Through the detection of 57 strains isolated from annual survey, no drug resistance mutations were found. Conclusions In this study,the A(H3N2) subtype influenza virus R292K and N294S mutation site TaqMan-MGB probe detection method bas been established. It is proved that the primer probe has high sensitivity and specificity with particular application potential.
Key words:  [Key words]H3N2 subtype influenza virus  Mutation  Realtime RT-PCR