体外6种疱疹类病毒DNA扩增及其基因分型

Amplification and detection of the six major Heperviruses by PCR-based assay

目的 快速检测 6种疱疹类病毒DNA。方法 在疱疹类病毒高度同源序列DNA多聚酶基因中设计两对通用引物 ,一对扩增单纯疱疹病毒Ⅰ型与Ⅱ型、爱泼斯坦 巴尔病毒和人巨细胞病毒 4种病毒 ,另一对扩增水痘 带状疱疹病毒和人类疱疹病毒 6型 2种病毒。经DNA聚合酶链反应(PCR)扩增 ,并对扩增产物进行分子克隆序列分析后 ,选择BamHⅠ或BstUⅠ酶切对扩增产物进行鉴别。结果 6种标准病毒株PCR扩增后产物为 5 10～ 5 92bp ,其最小检出量为 0 .1fg ,与乙型肝炎病毒、真菌、细菌和人类基因组DNA无交叉反应。用该方法检测临床 89份脑脊液 (CSF)和 75份血标本中的疱疹类病毒 ,13份 (14 .6 % )CSF和 2 6份 (34.7% )血标本为阳性 ,通过BamHⅠ或BstUⅠ两种酶切后能明确系何种疱疹类病毒。同时用酶联免疫吸附 (ELISA)法检测该标本的HSVⅠ /Ⅱ、EBV和CMV这 4种病毒的IgM ,10例阳性。这 4种病毒的PCR阳性检出率为 30 .7% ,而ELISA法为13.3% (P <0 .0 5 ) ,具有显著的差异性。 38例正常健康儿童的血和 9份非病毒感染的CSF标本 6种疱疹类病毒DNA均为阴性。结论 PCR加限制性内切酶片段长度多态性分析具有特异敏感 ,快速准确的特点 ,为疱疹类病毒感染的早期快速诊断提供了有效的手段。

Objective To detect and differentiate six major human herpesviruses DNA by PCR, RFLP, DNA clone and sequence analysis. Methods Based on the sequence of well coonserved regions of the DNA polymerse gene in human herpesvimses, we synthesized two pairs of primers, including one pair designed to amplify herpes simplex virus type 1 and 2, Epstein Barr virus and cytomegalovirus, other pair of primer to varicella zoster virus and human herpesvirus 6 by PCR. Identification of the virus species was achieved through restriction enzyme digestion with BamHI and BstUI. Results The products of six human herpesviruses after PCR amplification were from 510bp to 592bp and allowed characterization of herpesvirus type with restriction endonulease analysis. The sensitivity could reach 0.1fg DNA and had no cross reaction to human genomic DNA, bacteria, fungas and virus. 89 cerebrospinal fluids(CSF) and 75 blood specimens were tested for the presence of hepersvirus DNA by this PCR assay, in which 13(14.6%) CSF and 26(34.7%) blood specimens were positive. Comparatively, 10(13.3%) were positive by ELISA for HSVⅠ/Ⅱ,EBV,CMV IgM in blood specimens and significantly lower than that of the PCR rate (P<0.05). The hepersvirus type of these positive specimens was rapidly detected after restriction enzyme digestion with BamHI and BstUI. None of the 38 control blood and 9 control CSF specimens was positive for herpesvirus by this PCR. Conclusions This PCR-RFLP was specific, sensitive, rapid and accurate in providing a new technique for the identification of herpesvirus DNA in herpesvirus infection