One-step RT-PCR-RFLP方法筛查新型甲型H1N1流感病毒Oseltamivir耐药株

Screening Oseltamivir resistant strains in pandemic influenza A( H1N1) virus by a One-step RT-PCR / restriction fragment lengthpolymorphism assay

目的分析2013年江西省甲型H1N1流感病毒神经氨酸酶(NA)基因第275位氨基酸变异情况,掌握甲型H1N1流感病毒耐药特性,为甲型H1N1流感临床治疗和疾病控制提供参考。方法收集江西省流感监测哨点医院采集的流感样病例的鼻咽拭子标本,采用狗肾传代细胞(MDCK)对标本进行病毒分离,对分离的31株新型甲型H1N1流感病毒进行核酸提取,采用一步法逆转录-聚合酶链反应(One-step RT-PCR)扩增病毒NA基因部分片段,采用限制性内切酶片段长度多态性分析(RFLP)方法,分析扩增的部分NA基因第275位是否发生组氨酸(H)到酪氨酸(Y)的突变。结果2013年江西省31株甲型H1N1流感病毒NA基因第275位氨基酸均为组氨酸,未发生变异。结论 2013年31株毒株均对Oseltamivir敏感,One-step RT-PCR-RFLP方法筛查新型甲型H1N1流感病毒Oseltamivir耐药株简便可行。

Objective To analyze the characteristics of site 275 in NA protein of pandemic influenza H1N1 virus in Jiangxi province in 2013. Methods The specimens of nasopharyngeal swabs were collected from influenza like cases of influenza monitor hospitals and isolated for influenza virus with MDCK. All 31 strains of pandemic influenza A（ H1N1） 2009 virus were selected for detection and virus RNA were extracted. The specific gene fragment of NA genes were amplified by one-step RT-PCR and then were analyzed by restriction fragment length polymorphism（ RFLP） assay. Results All strains of pandemic H1N1 influenza virus had no mutation in position 275 of NA gene. Conclusion 31 strains were sensitive to Oseltamivir. The One-step RT-PCR-RFLP assay is simple and feasible for screening oseltamivir resistant pandemic influenza A（ H1N1）2009 virus.