应用PCR-RFLP技术检测中枢神经系统感染患儿6种疱疹类病毒DNA的实验研究

目的　建立一种能快速区分 6种疱疹类病毒DNA特异的限制性内切酶图谱。方法　 2 0 0 2年 1～ 12月浙江大学儿童医院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 ,该引物能同时扩增单纯疱疹病毒Ⅰ型与Ⅱ型 (HSVI/HSVⅡ )、爱泼斯坦—马尔病毒 (EBV)、人巨细胞病毒 (CMV)、水痘 带状疱疹病毒 (VZV)和人类疱疹病毒 6型 (HHV6) 6种病毒。选择BstUI内切酶对PCR产物进行酶切。 98例怀疑病毒性脑炎、脑膜炎患儿的脑脊液标本用PCR RFLP和ELISA法进行检测。结果　 6种标准病毒株PCR扩增后产物为 5 10～ 5 92bp ,经酶切后能区分为何种病毒 ,其最小检出量为 0 1fg ,与乙肝病毒、真菌、细菌和人类基因组无交叉反应。用该方法检测临床 98份脑脊液标本中的疱疹类病毒 ,13份阳性 ,同时用ELISA法检测该标本 ,5例阳性。PCR阳性检出率为 13 3% ,ELISA为 5 0 9% (P <0 0 5 )。 2例患儿在院外阿昔洛韦已治疗 2～ 3d ,但入院后在其脑脊液中仍能检测到病毒DNA。 10例诊断为疱疹病毒感染患儿经更昔洛韦每天 10mg/kg ,分两次静脉注射治疗 14d后 ,8例临床症状明显好转 ,2例病情未见好转。结论　建立的疱疹类病毒特异的限制性内切酶图谱具有特异敏感 ,快速准确的特点 ,且不受抗病毒药物的影响 ,为中枢神经系统疱

Detection of the six major human herpesviruses DNA for diagnosis of central nervous system infections in chil-dren with PCR-RFLP technique

Objective To detect and differentiate six major human herpesviruses DNA by PCR-RFLP.Methods Using the well-conserved regions of the DNA polymerase gene in human herpesviruses,we synthesized two pairs of primer,including one pair of primer designed to amplify herpes simplex virus type 1 and 2,Epstein-Barr virus and cytomegalovirus,another 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 BstUI.Results The products of six human herpesviruses after PCR amplification ranged from 510bp to 592bp and allowed characterization of herpesvirus type with restriction endonuclease analysis.The sensitivity could reach 0.1fg DNA and had no cross-reaction to human genomic DNA,bacteria,fungi and other viruses.Ninety-eight cerebrospinal fluids (CSF) were tested for the presence of hepersvirus DNA by this PCR assay,in which 13 CSF specimens were positive and positive rate was 13.3%,while 5 were positive by ELISA for viral antibody specificity index in CSF specimens and positive rate was 5.09%,which was significantly lower than that of the PCR rate (P<0.05).Eight cases were cured while two cases still uncured after 10-14 days of ganciclovir(GCV)therapy(10mg/kg of body weight twice daily).Conclusion The PCR RFLP is a specific,sensitive and accurate technique for the identification of herpesvirus DNA in suspected viral infection of central nervous system infections in children.