荧光定量PCR同步检测人类疱疹病毒6,7,8型方法的建立  

Development of Multiple Quantitative Fluorescent PCR for Detection of Human Herpesvirus-6,-7,-8

作　　者：郑毅 高应林[2] 张凡雄[2] 赵友云[2] 王业富[3] 唐景峰[3] 

机构地区：[1]湖北省中医药大学检验学院,湖北武汉430065 [2]湖北省中医院检验科,湖北武汉430061 [3]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

出　　处：《武汉大学学报（医学版）》2013年第1期9-13,共5页Medical Journal of Wuhan University

基　　金：国家科技部重大专项(编号:2009ZX10004-107)

摘　　要：目的:建立同步检测人类疱疹病毒6,7,8型(HHV-6,-7,-8)的多重荧光定量PCR方法。方法:分别针对HHV-6,-7,-8的U67、U36、ORF65基因设计3对引物和3种Taq Man-LNA探针,构建三重荧光定量PCR反应体系,用于同步扩增HHV-6,-7,-8。分别应用HHV-6,-7,-8质粒建立对应标准曲线,并对多重荧光定量PCR方法进行线性范围、灵敏度、特异性、重复性和扩增效率等方法学评价。以DNA测序法为参考方法,检测45例疑似HHV感染者外周血单个有核细胞(PBMC)中的HHV-6,-7,-8,对多重荧光定量PCR方法进行临床特异性和敏感性评估。结果:多重荧光定量PCR方法检测HHV-6,-7,-8的线性范围均在10~6-10^(11)Copies/L,相关系数均>0.99,灵敏度为8×10~5Copies/L,最低检测限为5×10~5Copies/L,无非特异性扩增,线性范围内HHV-6,-7,-8的批间、批内变异系数(CV)均<5.5%;与单重荧光定量PCR相比,检测HHV-6,-7,-8质粒含量的相关性分别为0.980,0.987,0.965。以DNA测序方法为金标准,多重荧光定量PCR检测HHV-6,-7,-8的特异性均为100%,灵敏度分别是93.1%、80%、100%。结论:多重荧光定量PCR能够对HHV-6,-7,-8进行同步、快速和准确的定量检测,将在输血安全筛查方面有着广泛的应用前景。Objective： To establish a multiplex fluorescence quantitative PCR method for simultaneous detection of human herpesvirus-6, -7, and 8 （HHV-6,-7,-8）. Methods： Three pairs of primers and TaqMan-LNA probes were selected by U67, U36, ORF65 of HHV-6 ,-7 ,-8 and they were used to construct a multiplex PCR system. Plasmids o{ HHV-6 ,-7 ,-8 were conducted to develop corresponding standard curves and to determine the dynamic range, sensitivity, specificity, and repetition of the method. Forty-five patients with suspected HHV-infection were quantitatively detected of HHV-6, -7, -8 with real-time FQ-PCR and DNA sequencing to evaluate the clinical specificity and sensitivity. Results： The quantitative range of multiplex PCR extended from 10^6-10^11 copies/L,r〉0.99, sensitivity were 8 × 10^5 copies/L, the limit of detection were 5 × 10^5 copies/L, and the intra and inter assay coefficient of variation were below 5.5 %. The correlation of multiplex PCR to single PCR was 0. 980, 0. 987, and 0. 965 for HHV-6 ,-7 ,-8 respectively. The specificity of multiplex FQ-PCR was all 100% for HHV-6 ,-7 ,-8 while the sensitivity was 93. 1%, 80%, and 100% respectively. Conclusion： The multiplex FQ-PCR was demonstrated as a rapid and sensitive method, for detecting HHV-6 ,-7 ,-8 and showed a great application prospect in blood screening.

关 键 词：人类疱疹病毒 多重荧光定量PCR TaqMan-LNA探针 输血筛查 

分 类 号：R752.1[医药卫生—皮肤病学与性病学]