利用实时聚合酶链反应检测人类正痘病毒感染及鉴别天花病毒

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time PCR.

作者信息

Putkuri Niina, Piiparinen Heli, Vaheri Antti, Vapalahti Olli

机构信息

Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.

出版信息

J Med Virol. 2009 Jan;81(1):146-52. doi: 10.1002/jmv.21385.

DOI:10.1002/jmv.21385

PMID:19031452

Abstract

We developed a real-time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real-time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 microl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections.

摘要

我们开发了一种实时聚合酶链反应（PCR）方法，用于从不同临床标本中检测正痘病毒（OPV），并将天花病毒与其他正痘病毒区分开来。在我们的方法中，我们使用自动核酸提取系统结合实时PCR，创建了一个简单、安全且快速的程序以获得初步结果。与使用SYBR Green I进行检测相比，使用设计的杂交探针时灵敏度更高。根据样本类型，每20微升反应体积的检测限为13至1300个拷贝。该PCR检测出了所有对人类致病的正痘病毒（天花、牛痘、猴痘、痘苗）以及骆驼痘病毒和鼠痘病毒。通过熔解曲线分析，可以将天花病毒序列的扩增与其他正痘病毒区分开来。我们还证明了该检测方法在人类牛痘和痘苗病毒感染病例中的适用性。