a Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India.
b School of Animal Biotechnology , Guru Angad Dev Veterinary and Animal Sciences University , Ludhiana , India.
Anim Biotechnol. 2017 Oct 2;28(4):248-252. doi: 10.1080/10495398.2016.1268620. Epub 2017 Jan 6.
Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (T) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.
牛疱疹病毒 1 型（BoHV-1）是一种重要的病毒病原体，给牛养殖业造成了重大的经济损失。糖蛋白 E 缺失标记疫苗是 BoHV-1 控制计划的基础，广泛应用于该病的防控，其中对野毒株和 gE 缺失疫苗株的检测和区分对于正确的疾病管理至关重要。本研究报告了一种基于 EvaGreen 的多重实时聚合酶链反应（EGRT-PCR）检测方法，可快速区分 BoHV-1 的野毒株和糖蛋白 E 缺失疫苗株。EGRT-PCR 检测方法可以在单个反应管中同时检测两个病毒基因（糖蛋白 B 和 E）和一个内参阳性对照基因（牛生长激素- bGH）。EGRT-PCR 检测方法的分析灵敏度低至每个反应 10 个 BoHV-1 DNA 拷贝。该改良的实时 PCR 检测方法可根据基因特异性解链温度（T）峰成功区分野毒株和 gE 缺失的 BoHV-1 株。我们的结果表明，本研究中开发的 EGRT-PCR 检测方法可能是一种有前途的疾病管理工具，可快速区分 BoHV-1 的野毒株和 gE 缺失疫苗株。
