聚合酶链反应-酶谱法分型检测人疱疹病毒DNA方法的建立及应用研究

POLYMERASE CHAIN REACTION-ENDONUCLEASE CLEAVAGE FOR THE DETEGTION OF HUMAN HERPESVIRUS DNA

在疱疹病毒高度同源序列DNA聚合酶基因中设计一对引物，能对HSV－Ⅰ、HSV－Ⅱ、EBV和CMV4种疱疹病毒DNA进行扩增，继而用凝胶电泳和限制性内切酶BarnHⅠ或SmaⅠ酶切分析扩增产物进行鉴别，建立了能一次性分型检测上述4种病毒的POR－酶谱法。敏感性和特异性试验表明，灵敏度可测到1fgDNA，相当于6个病毒颗粒，且引物仅对4种疱疹病毒扩增。对影响PCR效果的有关因素如Mg2＋浓度、引物浓度、循环参数等进行了优化选择。用建立的PCR－酶谱法检测病毒性脑炎患者脑脊液和慢性宫颈炎患者宫颈分泌物中的疱疹病毒，取得较好结果。本方法的建立为疱疹病毒感染的临床早期准确诊断、药物疗效考核和流行病学研究等提供了有效的手段。

A single pair of oligonucleotide primers selected within a highly conserved region of the DNA polymerase gene of the herpesvirus was designed to amplify the related viral genomes.i. e., herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus and cy-tomegalovirus, by the polymerase chain reaction(PCR). A simple restriction enzyme analysis of these amplified products allowed accurate characterization of the herpesvirus type. The specificity and sensitiyity of the PCR were asseooed. There was no cross-reaction with hu-man DNA or with DNA from othe pathogens. The lowest detecliou lcvel was lfg DNA,corresponding to apploximately the amount of DNA from 6 virions. The experimental condition were optimized including Mg2+ concentrations, primers concentration, temperature cycling protocols and method of DNA extraction. The results show that the PCR technqiue is highly sensitive and specific for the identification of herpesvirus and should be of value in the early and rapid diagnosis, therapeutic decisions, prognostic evaluation, and epidemiological studies.