荧光恒温扩增技术检测单纯疱疹病毒Ⅰ型RNA方法的建立  

Detection of herpes simplex virus Ⅰ RNA using simultaneous amplification and testing

作　　者：曹凌峰[1] 苏犁云[1] 施鹏[2] 董妞妞[1] 徐锦[1] 

机构地区：[1]复旦大学附属儿科医院临床检验中心,上海201102 [2]复旦大学附属儿科医院信息中心,上海201102

出　　处：《检验医学》2016年第3期163-167,共5页Laboratory Medicine

基　　金：上海市公共卫生优秀学科带头人计划(GWDTT201221)

摘　　要：目的利用荧光恒温扩增技术(SAT)建立一种快速、可靠的单纯疱疹病毒Ⅰ型(HSV-1)检测方法。方法根据Genbank上获取HSV-1开放读码框Us5区域的RNA序列设计扩增引物及优化探针,使用M-MLV反转录酶及T7 RNA多聚酶对HSV-1 RNA进行核酸扩增,同时利用荧光定量聚合酶链反应(PCR)仪进行实时荧光信号收集和检测。收集儿童病毒性脑炎或疑似患儿样本212份(脑脊液样本150份、血浆样本62份),以巢式PCR+测序为参比方法,对SAT结果进行Kappa一致性分析。结果 SAT检测HSV-1 RNA的灵敏度为100拷贝/μL,采用SAT检测212份临床样本,共检测出18例HSV-1阳性样本,巢式PCR检出16例阳性,经测序证实其中12例为阳性结果。SAT与巢式PCR+测序方法的Kappa值为0.785,其敏感性为100.0%、特异性为97.0%。结论建立的SAT检测脑脊液和血浆中HSV-1 RNA的方法具有敏感性高、特异性强、准确、快速、可靠的优点,适用于临床对单纯疱疹病毒性脑炎早期的快速诊断。Objective To establish a rapid and reliable simultaneous amplification and testing（SAT） for detecting herpes simplex virus Ⅰ（HSV-1）. Methods The specific primers were designed,and the probes were optimized according to RNA sequences in open reading frame Us5 region of HSV-1 from Gen Bank,and HSV-1 RNA was amplified by M-MLV reverse transcriptase and T7 RNA polymerase. The fluorescence signals were collected and determined by fluorescence quantitation polymerase chain reaction（PCR）. A total of 212 clinical specimens（150 cerebrospinal fluid specimens and 62 plasma specimens） were collected from child patients,who were diagnosed or suspected with viral meningitis,and they were determined. Using nested PCR plus sequencing as reference method,the Kappa consistency analysis was performed on the SAT results. Results The sensitivity of HSV-1 RNA by SAT was 100 copies/μL. A total of 18 specimens were positive by SAT, among which 16 specimens were positive by nested PCR,and 12 specimens were positive by sequencing. The Kappa value between SAT and nested PCR plus sequencing was 0.785,and the sensitivity and specificity were 100.0% and 97.0%,respectively. Conclusions The established SAT is a sensitive,specific,accurate,rapid and reliable method for detecting HSV-1 RNA in cerebrospinal fluid and plasma,and it should be a potential method in rapid diagnosis of herpes simplex encephalitis.

关 键 词：单纯疱疹病毒I型 荧光恒温扩增技术 单纯疱疹性脑炎 特异性 敏感性 

分 类 号：R446.1[医药卫生—诊断学]