非对称PCR-FP技术同步检测HSV-1/-2、CMV和EBV  

Simultaneous detection of four major human herpesviruses by asymmetric PCRfluorescence polarization assay

作　　者：张菊[1] 吴中亮[2] 李丁[1] 王香玲[3] 梁平[1] 郭宴海[1] 李宏伟[1] 颜真[1] 

机构地区：[1]第四军医大学药学系全军基因诊断技术研究所,西安710033 [2]第四军医大学西京医院神经内科,西安710032 [3]西安交通大学第二医院检验科,西安710004

出　　处：《临床检验杂志》2009年第6期405-407,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家高技术研究和发展计划(2008AA02Z444)

摘　　要：目的应用非对称PCR-荧光偏振（FP）技术建立一种同步检测全血中4种主要疱疹病毒（单纯疱疹病毒1／2型，巨细胞病毒、EB病毒）的新方法。方法以人疱疹病毒属通用引物行非对称PCR，扩增从179例样本中抽提的DNA。PCR扩增产物与特异性单纯疱疹病毒1／2型（HSV-1／-2）、巨细胞病毒（CMV）和EB病毒（EBV）寡核苷酸探针混合物温育杂交，荧光偏振检测技术检测杂交液荧光偏振值，据荧光偏振值判断病毒感染类型，并以DNA序列测定结果为参照。结果与DNA序列测定的阳性检测符合率为100％，但DNA序列测定检测均未检出多重混合感染。非对称PCR-FP方法对HSV-1／-2检测的灵敏度达到1．0×10^3拷贝／ml．对EBV和CMV检测的灵敏度达到2．0×10^3拷贝／ml。结论本法对于4种主要疱疹病毒感染的筛查及预防、预后判断具重要价值。Objective To establish a novel method to detect four major human herpesvirus simultaneously by fluorescence polarization （FP） assay based on asymmetric PCR. Methods A consensus primer system in human herpesvirus was used in an asymmetric PCR. The probes of herpes simplex virus types 1 and 2 （ HSV-1/-2 ）, cytomegalovirus （ CMV ）, and Epstein-Barr virus （ EBV ） labeled with different fluorophores were hybridized respectively with target amplicons. The infection was determined by the increased FP value. The DNA extracted from 179 samples was subjected to FP and sequence assay to evaluate the feasibility of this method. Results The minimum detection level of FP assay was 10 genome copies for HSV-1/-2,20 genome copies for both EBV and CMV. FP assay could detect confections more effectively than sequence assay. Conclusions A practical method was developed for the simultaneous detection of the four major human herpesviruses by FP assay based on an asymmetric PCR.

关 键 词：人疱疹病毒 荧光偏振 非对称PCR 同步检测 

分 类 号：Q446.9[生物学—生理学]