安徽蚌埠急性呼吸道感染患者咽拭子样本鼻病毒的分离与鉴定

摘要

目的 对安徽蚌埠急性呼吸道感染患者咽拭子样本进行人鼻病毒(human rhinovirus,HRV)的分离与鉴定.方法 选择安徽蚌埠哨点医院诊断为急性上呼吸道感染患者的108份咽拭子标本,提取病毒RNA,采用HRV通用引物进行荧光定量PCR检测.将阳性标本进行病毒分离,对HRV病毒株进行HRV VP1基因的扩增与进化树序列分析.结果 荧光定量PCR检测HRV呈阳性标本共5份,标本感染H 1-Hela细胞3代后只有1份标本出现显著细胞病变效应.此标本经RT-PCR扩增出HRV VP1基因,基因序列分析显示该病毒株与其他代表性HRV A型病毒株的VP1核苷酸同源性分别都在95％以上,命名为TYZQ201901,进化树显示该株病毒与RMH127/2 013株的遗传距离最近,在一个分支上,确认为HRV A型病毒.结论 本研究采用H1-Hela细胞顺利从安徽蚌埠急性呼吸道感染患者咽拭子样本分离到1株HRV A型病毒,初步建立了HRV病毒分离技术体系.%Objective To isolate and identify human rhinovirus (HRV) from throat swab samples from patients with acute respiratory infection in Anhui Bengbu.Methods The throat swab specimens from 108 patients with acute upper respiratory tract infection diagnosed in the Anhui Bengbu were selected as samples.RNA was extracted and detected by HRV universal primers;H1-Hela cells were infected with the positive samples for virus isolation,and HRV VP1 gene amplification was performed and gene phylogenetic tree analysis of the successfully isolated HRV strains was done.Results Only 5 samples were positive for HRV by the real-time PCR,and only one sample showed significant cytopathic effects after three passages of H1-Hela cells were infected,and the HRV VP1 gene was amplified by RT-PCR in the sample that was named TYZQ201901.The sequence analysis showed that the VP1 nucleotide homology of the strain with other representative HRV A strains was over 95％.The gene phylogenetic tree analysis showed that the strain had the closest genetic distance to the RMH127/2013 strain,and both were on a branch and was confirmed to be HRV type A virus.Conclusions An HRV type A strain virus was isolated from throat swab samples from patients with acute respiratory infection in Anhui Province by the H1-Hela cells,and the HRV virus separation technology system were initially established.