Abstract:

Objective To rapidly detect Japanese encephalitis virus (JEV) from Culex tritaeniorhynchus in Chengdu by real-time fluorescence reverse-transcriptase polymerase chain reaction (RT-PCR) assay, and to analyze the genotype of JEV. Methods Real-time fluorescence RT-PCR was used for detection of JEV infection. PrM segments of JEV from the nucleonic acid detected were amplified by one step RT-PCR, and were sequenced．Clustal X1.83 and Mega 5.0 were used to construct phylogenetic tree and analyze genotype and homology. Results A total of 10 656 mosquitoes of Cx. tritaeniorhynchus were collected with 219 pools in 2012. Seven pools of Cx. tritaeniorhynchus were positive for JEV at 3.2%. Based on sequence analysis, seven samples were identified to be genotypeⅠ JEV with the 674 bp sequence of PrM. Compared with some sequences of JEV GⅠ, the seven samples showed nucleotide and amino acid homologies of 91.5%-100% and 94.9%-100%, respectively, and were highly homology with the Sichuan strain of JEV (SC09-X08). Conclusion The prevalent JEV from Cx. tritaeniorhynchus in Chengdu was quickly detected by real-time fluorescence RT-PCR assay and confirmed to be genotypeⅠ.

Key words: Culex tritaeniorhynchus, Japanese encephalitis virus, Real-time fluorescence RT-PCR, Genotype, Homology analysis

摘要：

目的 从四川省成都地区采集三带喙库蚊样品, 快速检测蚊虫中的流行性乙型脑炎病毒(JEV), 并确定JEV基因型别。方法 采用实时荧光RT-PCR方法检测蚊体内的JEV。利用一步法RT-PCR试剂盒扩增阳性样品PrM区段特异性核苷酸序列, 测序后应用Clustal X1.83和Mega 5.0软件构建系统进化树, 并进行基因分型和同源性分析。结果 2012年采集三带喙库蚊雌蚊10 656只, 分为219份, 检测出7份JEV阳性核酸样品, 阳性率为3.2%。扩增PrM区域特异性核苷酸序列674 bp, 经鉴定均为基因Ⅰ型。与国内外部分GⅠ型的相关序列比较, 核苷酸同源性为91.5%～100%, 氨基酸同源性为94.9%～100%, 其中与四川省SC09-X08株同源性最高。结论 实时荧光RT-PCR法能快速检测三带喙库蚊中的JEV, 成都地区存在基因Ⅰ型JEV流行。

关键词: 三带喙库蚊, 流行性乙型脑炎病毒, 实时荧光RT-PCR, 基因分型, 同源性分析