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

Establishment of Detection Method for R292K and N294S Mutation Sites in H3N2 Influenza A Virus with TaqMan-MGB Probes

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realtimeRT-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（1000copies）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．

Objective To establish a detection method to R292K and N294S mutation sites in H3N2influenzaA virus with TaqMan-MGB probes.Method NA gene sequences of23A(H3N2)influenza strains wereacquired from NCBI database and lab isolations.According to sequence alignment results,specific TaqMan-MGBprobes and primers were designed for R292K and N294S mutation sites in multiple realtime PCR method.Thesensitivity was detected using the ten times dilution method.Clinical specimens,other subtypes of influenza andother common respiratory viruses were used to verify the specificity of the method.Results The designed probescould detect107(1000copies)dilution of the corresponding template.Other subtypes of influenza virus andcommon respiratory viruses showed no cross reaction.Through the detection of57strains isolated from annualsurvey,no drug resistance mutations were found.Conclusions In this study,the A(H3N2)subtype influenzavirus R292K and N294S mutation site TaqMan-MGB probe detection method bas been established.It is proved thatthe primer probe has high sensitivity and specificity with particular application potential.