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目的探讨一致-简并引物RT-PCR对手足口病患者粪便中肠道病毒的检测效果。方法根据Gen Bank中不同肠道病毒属病毒的多聚蛋白氨基酸序列,利用一致-简并引物(CODEHOP)设计程序设计合成1对简并引物,用一步RT-PCR法对20份手足口病患者粪便样本提取的RNA进行扩增,扩增产物测序后,用BLAST同源性比对初步确定基因型别,再与不同流行株进行遗传进化分析。结果 20份样本RT-PCR检测结果均为阳性,11份样本与肠道病毒EV71型(Enterovirus,EV71)同源性高达97%,与EV71病毒的6个流行株位于同一分支,遗传距离为0.031~0.037;7份样本与柯萨奇病毒A16型(Coxsackievirus,Cox A16)同源性高达99%,与Cox A16病毒的6个流行株位于同一分支,遗传距离为0.010~0.028;2份样本与Cox A10同源性高达99%,与Cox A10病毒的6个流行株位于同一分支,遗传距离为0.024~0.053。结论肠道病毒属一致-简并引物RT-PCR方法可以用于手足口病患者感染肠道病毒检测与分型。
Objective To investigate the detection effect of the consensus-degenerate primer RT-PCR on enterovirus in feces of hand-foot-mouth disease patients. Methods According to the amino acid sequence of polyprotein of different Enteroviruses in Gen Bank, a pair of degenerate primers was designed and synthesized by CODEHOP design protocol. The RNA extracted from the stool sample was amplified. After the amplified products were sequenced, the genotypes were determined by BLAST homology comparison, and the genetic evolution analysis was performed with different epidemic strains. Results The RT-PCR results of all the 20 samples were positive. The 11 samples were 97% identical to Enterovirus (EV71) EV71 and were located on the same branch with 6 strains of EV71 EV71, with a genetic distance of 0.031 ~ 0.037. The homology between 7 samples and Coxsackievirus A16 (Cox A16) was as high as 99%, which was located on the same branch with 6 strains of Cox A16 virus, with a genetic distance of 0.010 ~ 0.028. Two samples The homology of Cox A10 was as high as 99%. It was located on the same branch with 6 epidemic strains of Cox A10 and the genetic distance was 0.024 ~ 0.053. Conclusions The enteroviruses are identical - the degenerate primer RT-PCR method can be used for the detection and typing of enterovirus in hand, foot and mouth disease patients.