摘要
目的建立一种灵敏且特异的实时荧光定量RT-PCR方法,用于快速检测盖塔病毒(getah virus, GETV)。方法从GenBank数据库下载GETV基因序列,使用Clustal X完成序列比对,针对高保守区段设计特异性引物和探针;以GETV核酸为标准品建立标准曲线,分别对检测反应的灵敏度、特异性和稳定性进行评价。结果本方法能特异地检测GETV,与多种虫媒病毒无交叉反应;灵敏度为1.0×10 pfu/ml,批内和批间变异系数均小于1%。从湖南、河北、福建、重庆采集的196批蚊虫中检出1批GETV阳性。结论建立了灵敏度高,特异性强的TaqMan探针实时荧光定量RT-PCR方法用于GETV筛查。
Objective To establish a sensitive and specific real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)method for rapid detection of Getah virus(GETV).Methods All the gene sequences of GETV were downloaded from GenBank database.Clustal X was used for sequence alignment,and specific primers and probes were designed according to highly conserved regions;we established a standard curve using the nucleic acid of GETV as a standard,and the sensitivity,specificity and stability of this method were evaluated respectively.Results This method could specifically detect GETV and has no cross-reactivity with multiple arboviruses;the sensitivity was 1.0×10 pfu/ml,and the intra-assay and inter-assay coefficients of variation were less than 1%.One case was GETV positive in 196 batches of mosquitoes collected from Hunan province,Hebei province,Fujian province and Chongqing city.Conclusions We established a TaqMan probe real-time quantitative RT-PCR with high sensitivity and specificity which can be used for screening.
作者
吴天元
付士红
殷启凯
赵洁荣
李樊
何英
许松涛
梁国栋
聂凯
杨光
王环宇
Wu Tianyuan;Fu Shihong;Yin Qikai;Zhao Jierong;Li Fan;He Ying;Xu Songtao;Liang Guodong;Nie Kai;Yang Guang;Wang Huanyu(School of Medicine,Jinan University,Guangzhou 510000,China;National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China.)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2021年第2期205-208,共4页
Chinese Journal of Experimental and Clinical Virology
基金
国家科技重大专项(2018ZX10711001)
国家重点研发(2017YFC1200505)
传染病预防控制国家重点实验室重点项目(2015SKLID505)。
