2种RT-PCR技术检测肠道病毒RNA的对比研究

Comparison of two different PCR assays for detection of enterovirus RNA in central nervous system

目的 比较 2种逆转录多聚酶链反应 (RT PCR)技术检测脑脊液中肠道病毒 (EV)RNA。方法 设计与肠道病毒基因组保守的 5′非编码区同源的引物。方法A :采用AMV逆转录酶和TaqDNA聚合酶 ,逆转录合成cDNA与PCR在不同缓冲体系中分别完成 ;方法B :AMV逆转录酶和TflDNA聚合酶同时加入 ,逆转录与PCR在同一缓冲体系中进行 ,逆转录后不打开反应管。结果 通过检测不同稀释度的PV1、CVB3和CVB4病毒及无菌性脑膜炎病人CSF中EVRNA ,证明 2种方法有相同的敏感性 ,而方法B在逆转录后不需打开反应管 ,降低了污染的危险 ,更便于自动化操作。结论 2种RT PCR技术均是检测EV感染的有效方法 。

Objective To compare two reverse transcription polymerase chain reaction (RT-PCR) assays which were developed to detect enteroviral RNA in cerebrospinal fluid (CSF) samples.Methods Primers homologous to the conserved 5′noncoding region of the enterovirus genome were designed.The RT-PCR product size was 194bp.In assay A AMV reverse trascriptase were utilized for reverse transcription,Taq DNA polymerase for subsequent PCR and the reactions were completed in different experimental systems.In assay B AMV reverse transcriptase and Tfl DNA polymerase were utilized and the reactions were completed in the same buffer.After reverse transcription step the reaction tubes was not opened.Result By detecting polioviral I,coxackievirus B3 and B4 with serial dilutions and enteroviral RNA in CSF samples from patients with acute aseptic meningitis,it was demonstrated the two assays had similar sensitivity,but in assay B opening the reaction tube was avoided so the risk of contamination was reduced.Conclutions Both of the two RT-PCR methods are effective for diagnosis of enterovirus infection in central nervous system.