实时荧光定量PCR鉴别检测单纯疱疹病毒方法的建立  

Establishment of Real-Time FQ-PCR for Quantitative Detection and Differentiation of Herpes Simplex Virus

作　　者：赵友云[1] 周立[1] 高应林[2] 王业富[1] 

机构地区：[1]武汉大学生命科学学院/病毒学国家重点实验室,湖北武汉430072 [2]湖北省中医院检验科,湖北武汉430061

出　　处：《武汉大学学报（医学版）》2008年第5期617-619,共3页Medical Journal of Wuhan University

摘　　要：目的:建立快捷、灵敏、特异的单纯疱疹病毒(HSV)的实时荧光定量聚合酶链反应(FQ-PCR)检测及分型方法。方法:选择HSV1和HSV2的糖蛋白B基因设计一对通用引物和对应的探针组建2个FQ-PCR反应体系,应用克隆的质粒建立系列标准品制作标准曲线,进行重复性和敏感性试验,以其他种类的细菌、病毒、真菌进行特异性分析,并用于检测生殖器疱疹患者的HSV1和HSV2。结果:HSV1和HSV2两标准曲线的线性检测范围均为105到1011Copies/L,相关系数均大于0.99,其他种类的细菌、病毒、真菌均未出现特异性扩增;48例生殖器疱疹患者疱液中HSV1和HSV2的检出率分别为6.3%(3/48)和87.5%(42/48)。结论:实时FQ-PCR法鉴别检测HSV1和HSV2,具有快速、特异、灵敏、便捷的特点,有助于HSV感染的流行病学和病理机制的研究;生殖器疱疹患者以HSV2感染为主。Objective： To establish a rapid,sensitive and specific real time fluorescent quanlitative polymerase chain reaction（FQ-PCR） method for quantitative detection and differentiation of herpes simplex virus（HSV）. Methods： Primers and TaqMan probes were selected by comparing the glycoprotein B gene sequence of HSV1 and HSV2. Plasmids containing target fragments of HSV1 and HSV2 were conducted as the external standards to develop the real time FQ-PCR and to de termine the repetition and sensitivity of each assay. Additional reference strains from the bacteria,virus and epiphyte were used to test the specificity of primers and probes, and forty clinical specimens were quantitative detected for differentiation of HSV1 and HSV2 with real time FQ- PCR. Results： The quantitative range of the assays extended from 10s-10u copies of each liter, r 〉0.99,and specificity was determined by non-amplification different human pathogens,including bacteria,virus and epiphyte. Application of this assay to 48 clinical specimens collectes from pa- tients with genital herpes. Their detection rates were 6. 3% for HSV1（3/48） and 87. 5% for HSV2（42/48）. Conclusion： This real-time FQ-PCR was demonstrated as a rapid, specific,sensitive and convenient method for quantitative detection and differentiation of HSV1 and HSV2,it is helpful in the study of the pathogenesis and epidemiology of HSV. The patients with genital herpes were mostly infected by HSV2.

关 键 词：疱疹 生殖器 HSV1 HSV2 实时荧光定量PCR 

分 类 号：R311[医药卫生—基础医学]