四重荧光定量rRT-PCR法检测柯萨奇病毒B1、B2、B3及其他未分型肠道病毒  

Four fluorescent quantitative real-time RT-PCR assay for detection of coxsackievirus and universal enterovirus

作　　者：龚建光[1] 崔大伟[1] 谢国良[1] 杨先知[1] 郭希超[1] 陈瑜[1] 

机构地区：[1]浙江大学医学院附属第一医院检验科,杭州310003

出　　处：《临床检验杂志》2013年第8期561-564,共4页Chinese Journal of Clinical Laboratory Science

基　　金："十二五"国家科技重大专项(2012ZX10004-210);浙江省教育厅资助项目(Y201226071)

摘　　要：目的建立检测柯萨奇病毒(CoxB1、CoxB2、CoxB3)和其他未分型肠道病毒(EV)的四重荧光定量rRT-PCR法。方法用Primer Express 3.0软件设计特异性引物和探针,优化反应体系,评价建立的rRT-PCR法的灵敏度、特异性和重复性,并用该法检测535份病毒性心肌炎患儿粪便标本和43份咽拭子标本,对阳性结果测序验证。结果 rRT-PCR法检测CoxB1、CoxB2、CoxB3和其他未分型EV灵敏度均达103copies/mL,特异性较高,检测CV均<1.0%。578份临床标本用该法检测EV为153份,其中CoxB1病毒69份,CoxB2病毒18份,CoxB3病毒27份,其他未分型EV 39份。随机选取CoxB1、CoxB2、CoxB3病毒检测阳性样本各10例测序,均与该法的检测结果一致。结论建立的四重荧光定量rRT-PCR法可同时检测并区分CoxB1、CoxB2、CoxB3病毒和其他未分型EV,可用于此类病毒引起的暴发疫情的实验室应急诊断。Objective To establish a four fluorescent quantitative real-time reverse transcription-PCR （rRT-PCR） assay for detection of coxsaekievirus B1 （CoxB1） , B2, B3 and universal enterovirus simultaneously. Methods We designed the specific primers and probes of Cox B1, B2, 133 and universal enterovirus for PCR by using Primer Express 3.0 software and optimized the reaction system. The sensitivity, specificity and repeatability of the assay were evaluated. A total of 535 feces specimens and 43 pharyngeal swab speci- mens from the children with viral myocarditis were detected by the developed method and the positive results were verified by sequen- cing. Results The sensitivities of the assay reached to 103 eopies/mL. The assay showed high specificity and the coefficients of varia- tion were all less than 1.0%. Of the 578 clinical specimens, 153 specimens were positively detectable by the four rRT-PCR, including 69 CoxB1, 18 CoxB2, 27 CoxB3 and 39 other subtypes of enterovirus. Randomly assigned 10 specimens from each group of CoxB1, B2 or B3-positive results by the established assay were sequenced and consistent results were obtained. Conclusion The established four rRT-PCR assay may detect CoxB1, CoxB2 and CoxB3 virus and universal enterovirus in a single test tube. The assay should be valuable for clinical diagnosis of epidemic outbreak of infection caused by CoxB1, B2, B3 viruses and universal enterovirus.

关 键 词：肠道病毒 柯萨奇病毒 RRT-PCR 

分 类 号：R446.5[医药卫生—诊断学]