通用引物聚合酶链反应一次检测4种疱疹病毒DNA

Detection of human herpesvirus DNA by the polymerase cbain reaction-endonuclease cleavage analysis

为了能一次性分型检测４种疱疹病毒ＤＮＡ，防止漏诊，故采用在疱疹病毒高度同源序列ＤＮＡ聚合酶基因中设计一对通用引物的方法，对４种疱疹病毒（单纯疱疹病毒Ⅰ型、Ⅱ型、爱泼斯坦巴尔病毒、人巨细胞病毒）ＤＮＡ进行扩增，继而用凝胶电泳和限制性内切酶ＢａｍＨＩ或ＳｍａＩ酶切扩增产物进行鉴别，建立了能一次性分型检测上述４种病毒的聚合酶链反应（ＰＣＲ）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。用建立的ＰＣＲ法和酶切分型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。该法的建立为疱疹病毒感染的临床早期准确诊断、判定药物疗效和流行病学研究等提供了有效的手段。

single pair of oligonucleotide primers selected within a highly conserved region of the DNA poly-merase gene of the herpesvirus was designed to amplify related viral genomets,i.e.,herpes simplex virus type 1,herpes simplex virus type 2,Epstein-Barr virus,and cytomegalovirus,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 sensitivity of this PCR-RFLPs were exam-ined.There was no cross-reaction with human DNA or with DNA from other pathogens.The lowest detection level was 1fg DNA,corresponding approximately to the total amount of DNA from 6 virions. The results show that the PCR-RFLPS provides a highly sensitive and specific technique for the identifi-cation of herpesvirus infection and it should be of value for early and rapid diagnosis,therapeutic and prognostic evaluations and epidemiological studies.