Real-time PCR to identify variola virus or other human pathogenic orthopox viruses
Real-time PCR to identify variola virus or other human pathogenic orthopox viruses
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DOI：
10.1373/clinchem.2006.068635
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发表时间：
2007-04-01
影响因子：
9.3
通讯作者：
Garin, Daniel
中科院分区：
文献类型：
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作者：
Scaramozzino, Natale;Ferrier-Rembert, Audrey;Garin, Daniel
背景：天花病毒（痘病毒科，正痘病毒属）和密切相关的牛痘、牛痘和猴痘病毒可感染人类。由于担心天花病毒可能被用作生物恐怖主义的武器，正在加紧努力补充天花疫苗库存，优化痘病毒的诊断方法，并开发新的天花抗病毒药物。方法：建立了一种检测天花病毒DNA的方法。该检测基于TaqMan化学靶向14kd蛋白基因。在实验的第一阶段，我们使用genu的共识引物和2个探针（14-kD POX和14-kD VAR）的混合物，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探针14-kD POX检测阳性样本，以鉴定猴痘、牛痘和牛痘病毒，并用14-kD VAR探针检测天花病毒。在4种不同的PCR循环器平台上建立了该实验。在一项85种不同的正痘病毒种类和毒株的研究中对其进行了评估，其中包括天花病毒、骆驼痘病毒、牛痘病毒、猴痘病毒和牛痘病毒，浓度范围为100 ng/L至1 μ g/L。结果：该方法仅检测到0.05 fg的DNA，相当于25个DNA拷贝，并使天花病毒与其他正痘病毒区分开来。结论：该实时荧光定量PCR技术为天花和其他人类致病性正痘病毒感染的早期检测和鉴别提供了一种快速的方法。(c) 2007美国临床化学协会。
Background: 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.Methods: 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 genu's 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 mu g/L.Results: 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.Conclusions: This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections. (c) 2007 American Association for Clinical Chemistry.