目的建立同时检测柯萨奇病毒A2和A5型（CVA2、CVA5）的双重实时荧光定量RT-PCR法。方法用Primer Express3.0软件设计高度特异性引物和荧光标记探针。通过优化反应体系,建立标准曲线,并评价双重荧光定量RT-PCR法的特异性、灵敏度和重复性。用建立的方法检测367例疑似手足口病患儿的粪便标本,并对阳性结果测序验证。结果荧光定量RT-PCR结果表明本实验所选的引物和探针可特异性检测并区分出CVA2、CVA5亚型,与其他肠道病毒的阳性核酸未发生交叉反应。该方法最低检测限达到10^2copies/m L,批内变异系数（CV）均〈1.49%。367份临床标本中CVA2型阳性23份,阳性率6.3%;CVA5阳性11份,阳性率3%,检测阳性结果与测序结果一致。结论成功建立了在单管中同时检测并鉴别CVA2、CVA5型的双重实时荧光定量RT-PCR检测方法。该方法具有较好的灵敏度、特异性、重复性,可用于CVA2、CVA5型引起的暴发疫情的快速筛查和手足口病的流行病学的研究。
Objective To establish a double fluorescent quantitative RT-PCR assay for simultaneously detecting coxsackievirus A2（ CVA2） and A5（ CVA5） subtypes. Methods The specific primers and probes of CVA2 and CVA5 subtypes for fluorescent quantitative RT-PCR assay were designed with Primer Express 3. 0 software. The optimizing double fluorescent quantitative RT-PCR system with a standard curve was established,and its specificity,sensitivity and repeatability were evaluated. Then,the stool specimens from367 children suspected with hand-foot-and-mouth disease were detected by this assay,and the positive specimens were further sequenced. Results The established double fluorescent quantitative RT-PCR was able to detect CVA2 and CVA5 subtypes specifically,and had no cross reaction with other enteric viruses. Its lowest detectable limit was 102 copies / m L,and the intra assay coefficients of variation were all less than 1. 49%. The positive rates of CVA2 and CVA5 subtypes in 367 clinical specimens were 6. 3%（ 23 /367）and 3%（ 11 /367）,respectively,and the positive results were verified by subsequent sequencing. Conclusion The double fluorescent quantitative RT-PCR assay for simultaneously detecting CVA2 and CVA5 subtypes in a single test tube is successfully established,which has good specificity,sensitivity and repeatability,and may be used to rapidly screen the infections caused by CVA2 and CVA5 subtypes and investigate the epidemiology of hand-foot-and-mouth disease.
Chinese Journal of Clinical Laboratory Science
fluorescent quantitative RT-PCR
coxsackievirus A2 subtype
coxsackie virus A5 subtype