中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (5): 273-275.

• 论著 • 上一篇    下一篇

荧光多重实时PCR检测单纯疱疹病毒

赖维1, Cheng-Yen Chen2, 苏向阳1, 黄怀球1, 张玉清1, Ron C Ballard2, Stephen A Morse2   

  1. 1. 中山大学附属第三医院皮肤性病科, 广州510630;
    2. 美国疾病控制与预防中心
  • 收稿日期:2003-05-20 出版日期:2004-05-15 发布日期:2004-05-15

Multiplex Fluorescent Real-time PCR for Detection and Typing of Herpes Simplex Virus

LAI Wei1, Cheng-Yen Chen2, SU Xiang-yang1, HUANG Huai-qiu1, ZHANG Yu-qing1, Ron C Ballard2, Stephen A Morse2   

  1. Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
  • Received:2003-05-20 Online:2004-05-15 Published:2004-05-15

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摘要： 目的 建立单纯疱疹病毒的荧光多重实时PCR检测及分型技术,为生殖器疱疹的诊断及病毒分型提供快捷、敏感、特异的方法。方法 以细胞培养法为对照,用Smart Cycler® System作为实验平台,以DNA探针荧光标记技术建立荧光多重实时PCR,用于检测生殖器疱疹患者临床标本中的1型和2型单纯疱疹病毒(HSV1、HSV2)DNA。结果 荧光多重实时PCR检测HSV1和HSV2DNA的敏感性和特异性均为100%,可检测出低至1~5拷贝的HSVDNA,30min左右即可完成整个检测过程,而细胞培养的敏感性和特异性分别为70.10%和100%。结论 基于Smart Cycler® System的荧光多重实时PCR是一种易于操作、扩增效率高、封闭式的核酸扩增技术,可用一次PCR反应就能快速、敏感、准确地对临床标本中的单纯疱疹病毒进行检测和分型。

关键词: 疱疹病毒1型,人, 疱疹病毒2型,人, 聚合酶链反应, 疱疹,生殖器

Abstract: Objective To develop a multiplex fluorescent real-time PCR method and typing technique of herpes simplex virus for providing a rapid tool with a high sensitivity and specificity for the diagnosis of genital herpes and HSV typing. Methods By using the viral culture as a standard control, the Smart Cycler® System as a laboratory platform and the fluorescence-labeled probe technique was used to develop a multiplex fluorescent real-time PCR for the detection of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA in clinical specimens of patients with genital herpes. Results Both sensitivity and specificity of the multiplex fluorescent real-time PCR in detecting HSV-1 and HSV-2 DNA were 100%, whereas the sensitivity and specificity of viral culture were 70.1% and 100%, respectively. It took about 30 minutes to complete the whole process with the multiplex real-time PCR, and the detectable limit was as low as 1-5 copies of HSV DNA. Conclusions The multiplex fluorescent real-time PCR based on Smart Cycler® System is an easy-to-operate and closed amplification system with high amplification efficacy. A single PCR test can provide a rapid, sensitive and accurate detection and typing of HSV in clinical practice.

Key words: Herpes genitalis, Herpesvirus 1, human, Herpesvirus 2, human, Polymerase chain reaction