通用引物检测儿童血和脑脊液的6种疱疹类病毒

Determination of six major human herpesviruses in cerebrospinal fluid and blood of children with consensus primers

目的 :通过 PCR- RFLP、DNA克隆和测序分析来检测和区分 6种疱疹类病毒 ,探讨其在临床上的应用价值。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 1型和 2型、爱泼斯坦 -马尔病毒、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 PCR扩增 ,并对扩增产物进行分子克隆、序列分析后 ,选择 Bam H 或 Bst U 酶切扩增产物进行鉴别。最后 ,对临床 38份脑脊液 (CSF,临床诊断为病毒性脑炎的病例 )和 4 9份血标本 (其中 2 7份为确诊病例 ,2 2份为临床诊断病例 )进行疱疹病毒的检测。结果 :38份 CSF标本中 13份阳性 (34.2 % ) ,2 7份确诊病例标本均阳性 (10 0 % ) ,2 2份临床诊断病例标本 16份阳性 (72 .7% )。这些阳性标本通过 Bam H 或 Bst U 两种酶切后能明确系何种疱疹类病毒。 6 8例正常健康儿童的血和 9份非病毒感染的脑脊液标本 6种疱疹类病毒 DNA均为阴性。结论 :聚合酶链反应加限制性内切酶片段长度多态性分析为疱疹类病毒感染的临床诊断提供了一种有效的手段。

Objective: To identify 6 major human herpesviruses with consensus primers and to explore its clinical application. WTHZ Methods: Based on the highly homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1,type 2,Epstein Barr virus and cytomegalovirus; and another was used to amplify varicella zoster virus or human herpesvirus 6.Virus species identification was performed by restriction enzyme digestion with BamH Ⅰ and BstU Ⅰ.Thirty eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for hepersvirus DNA using the PCR RFLP assay with these primers. Results: Thirteen out of 38 CSF specimens (34.2%) were hepersvirus positive.All blood specimens from 27 confirmed cases showed positive results,while for 22 clinically diagnosed cases 16 (72.7%) were positive.The types of hepersvirus were determined using restriction enzyme digestion with BamH Ⅰ and BstU Ⅰ.Two CSF specimens from the patients, who were treated with aciclovir for 2~3 days, were still positive for hepervirus DNA by this method.None of the control blood or CSF controls were positive for herpesvirus by PCR. Conclusion: The PCR RFLP method used in this study is a specific,sensitive and practicable one for diagnosis of herpesvirus infection.