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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
Objective To establish a DNA specific restriction endonuclease map that can quickly distinguish six kinds of herpesviruses. Methods From January to December 2002, two pairs of universal primers were designed in Children’s Hospital of Zhejiang University for highly homologous DNA polymerase gene of herpesvirus. The primers can simultaneously amplify herpes simplex virus type I and type II (HSVI / HSV II ), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), varicella-zoster virus (VZV) and human herpesvirus 6 (HHV6) The BstUI endonuclease was chosen to digest the PCR product. CSF samples of 98 children with suspected viral encephalitis and meningitis were tested by PCR RFLP and ELISA. Results The products of PCR amplification of 6 standard strains were 510 ~ 5 92bp. After being digested, they could be distinguished from each other by a minimum detectable amount of 0 1fg. There was no cross reaction with HBV, fungi, bacteria and human genome . The method was used to detect herpesviruses in 98 samples of cerebrospinal fluid in cerebrospinal fluid samples, and 13 samples were positive. Meanwhile, 5 samples were positive by ELISA. PCR positive detection rate of 13 3%, ELISA was 59% (P <0 05). Two patients were treated with acyclovir 2 to 3 days outside the hospital, but viral DNA was still detected in their cerebrospinal fluid after admission. 10 cases diagnosed as herpes virus infection in children with ganciclovir daily 10mg / kg, intravenous injection of two 14d, the clinical symptoms of 8 patients improved significantly, 2 patients did not improve the condition. Conclusion The herpes virus-specific restriction enzyme map is sensitive, rapid and accurate, and is not affected by antiviral drugs. It is the central nervous system blister