应用逆转录聚合酶链反应检测肠道病毒感染

Dection of enterovirus infection by reverse transcription and polymerase chain reaction

为建立一种能检测出临床上绝大多数肠道病毒（ＥＶ）感染的快速、敏感、特异的逆转录聚合酶链反应（ＲＴ－ＰＣＲ）方法，通过对ＥＶ基因组的序列分析，在其５′端非编码区设计一对引物，用该引物建立的ＲＴ－ＰＣＲ方法对３１种ＥＶ标准毒株和３４例无菌性脑膜炎及１１例无菌性脑炎患儿的脑脊液（ＣＳＦ）分别进行扩增，其产物做琼脂糖凝胶电泳及斑点杂交检测。研究表明，３１种ＥＶ标准毒株斑点杂交的敏感度达１０－２～１０－３５０％组织培养感染量（ＴＣＩＤ５０）。３４例无菌性脑膜炎中２１例阳性（６１．８％），１１例无菌性脑炎中８例阳性（７２．７％）；阳性患儿恢复期ＣＳＦ，２例无菌性脑膜炎和１例无菌性脑炎仍为阳性。提示用ＲＴ－ＰＣＲ方法检测ＥＶ感染是值得推广的。

To establish a rapid diagnostic method for enteroviral infections in clinic, the authors developed a reverse transcription and polymerase chain reaction (RT PCR) assay. Primers homologous to the conserved 5′ noncoding region were designed by analysis of enteroviral genomes, and then were used to enzymatically amplify enterovirus (EV) specific RNA fragment from 31 prototype enteroviral strains and cerebrospinal fluid (CSF) of 34 patients with aseptic meningitis and 11 patients with encephalitis of unknown etiology. The amplified products were detected by agar gel electrophoresis and slot blot hybridization analysis. The study on sensitivity of the RT PCR showed that the amplification could detect EV RNA from 10 -2 ～10 -3 50% tissue culture infective doses of the cultured viral strains. The results also showed that 21 (61.8%) of 34 aseptic meningitis cases and 8 (72.7%) of 11 encephalitis cases were positive for EV RNA in CSF samples. Two meningitis and one encephalitis cases were positive during convalescence. These results suggested that this RT PCR method was rapid, sensitive and specific in detection of most common EV infections.