Scaramozzino Natale, Ferrier-Rembert Audrey, Favier Anne-Laure, Rothlisberger Corinne, Richard Stéphane, Crance Jean-Marc, Meyer Hermann, Garin Daniel
Laboratoire de Virologie, Centre de Recherches du Service de Santé des Armées (CRSSA) Emile Pardé, Grenoble, France.
Clin Chem. 2007 Apr;53(4):606-13. doi: 10.1373/clinchem.2006.068635. Epub 2007 Mar 1.
Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism.
We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L.
The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses.
This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.
天花病毒（痘病毒科正痘病毒属）以及与之密切相关的牛痘病毒、痘苗病毒和猴痘病毒均可感染人类。由于担心天花病毒可能被用作生物恐怖主义武器，补充天花疫苗储备、优化痘病毒诊断方法以及研发新型抗天花病毒药物的工作正在加紧进行。
我们开发了一种检测天花病毒DNA的检测方法。该检测方法基于针对14-kD蛋白基因的TaqMan化学方法。在检测的第一阶段，我们使用属特异性引物和两种探针（14-kD POX和14-kD VAR）的混合物，该混合物跨越14-kD蛋白编码基因，用于检测所有人类致病性正痘病毒。然后，我们用特异性正痘病毒特异性探针14-kD POX检测阳性样本，以鉴定猴痘病毒、牛痘病毒和痘苗病毒，并用14-kD VAR探针鉴定天花病毒。该检测方法在4种不同的PCR循环仪平台上建立。在一项研究中，我们用85种不同的正痘病毒种类和毒株对其进行了评估，这些毒株包括天花病毒、骆驼痘病毒、牛痘病毒、猴痘病毒和痘苗病毒，浓度范围为100 ng/L至1 μg/L。
该检测方法能检测低至0.05 fg的DNA，相当于25个DNA拷贝，并能区分天花病毒与其他正痘病毒。
这种实时PCR检测方法为早期检测和区分天花及其他人类致病性正痘病毒感染提供了一种快速方法。
