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首页> 外文期刊>Journal of Virological Methods >Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1.
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Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1.

机译：快速检测和区分野生型和糖蛋白E缺失的1型牛疱疹病毒疫苗株的三重实时PCR检测方法的开发和验证。

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摘要

Two important components of control programs for Bovine herpesvirus type 1 (BoHV-1), a major pathogen of cattle, are the detection of outbreaks and vaccination with glycoprotein E (gE)-deleted marker vaccines. In addition to serology, rapid and accurate investigation of BoHV-1 and genetic differentiation of vaccine and wild-type strains are also important methods. Therefore, a triplex quantitative real-time polymerase chain reaction (qPCR) for testing BoHV-1 was developed. Apart from a BoHV-1 specific glycoprotein D (gD) assay, a gE-specific system for differentiation between wild-type BoHV-1 and gE-deleted vaccine strains was established. Finally, an internal control, based on the beta-actin gene was introduced successfully completing the multiplex system. The triplex BoHV-1-qPCR has an analytical sensitivity of less than 10 genome copies per reaction, and the diagnostic sensitivity was equal to or even greater than that of the 'gold standard' method of virus isolation in cell culture. A series of reference strains, including gE-deleted BoHV-1 and field isolates were detected reliably. For validation of the specificity of the test, nasal swabs, semen and different organ material from cattle, negative for BoHV-1, and genetically related herpesvirus strains were examined. The new multiplex BoHV-1-specific qPCR system allows highly sensitive and rapid genetic detection and differentiation of BoHV-1 and will be a valuable method for the control of BoHV-1 infection.
机译：牛主要病原体 1型牛疱疹病毒（BoHV-1），控制程序的两个重要组成部分是检测暴发和接种糖蛋白E（gE）缺失标记疫苗。除血清学外，对BoHV-1进行快速，准确的研究以及疫苗和野生型菌株的遗传分化也是重要的方法。因此，开发了用于测试BoHV-1的三重实时定量聚合酶链反应（qPCR）。除了BoHV-1特异性糖蛋白D（gD）分析外，还建立了用于区分野生型BoHV-1和gE缺失疫苗株的gE特异性系统。最后，成功引入了基于β-肌动蛋白基因的内部对照，完善了多重系统。三重BoHV-1-qPCR的每个反应分析灵敏度低于10个基因组拷贝，诊断灵敏度等于或什至大于细胞培养中病毒分离的“金标准”方法。可靠地检测了一系列参考菌株，包括缺失gE的BoHV-1和现场分离株。为了验证测试的特异性，检查了牛的鼻拭子，精液和不同器官的材料，BoHV-1阴性以及遗传相关的疱疹病毒株。新的多重BoHV-1特异性qPCR多重系统可实现BoHV-1的高度灵敏，快速的基因检测和分化，将是控制BoHV-1感染的有价值的方法。

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