El-Kholy Alaa A, Abdelrahman Khaled, Soliman Hatem
Veterinary Serum & Vaccine Research Institute, Abbassia, P.O. Box # 131, Cairo, Egypt.
Parasitology and Animal Diseases Department, National Research Center, Dokki, Giza, Egypt.
J Virol Methods. 2014 Aug;204:81-5. doi: 10.1016/j.jviromet.2014.04.011. Epub 2014 Apr 24.
Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65°C for 60min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1fg of BoHV-1 DNA (dilution of 10(6)). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt.
牛疱疹病毒1型（BoHV-1）是家养和野生牛的一种严重病毒病原体。在此，我们报告了一种利用环介导等温扩增（LAMP）技术快速灵敏检测BoHV-1的新型分子诊断方法的开发。BoHV-1-LAMP检测方法经过优化，通过将Bst-DNA聚合酶与一组基于BoHV-1病毒gE基因特别构建的六条引物在65°C孵育60分钟来扩增目标DNA。BoHV-1-LAMP产物通过使用SYBR Green-I染色进行目视检查来检测，并且通过凝胶电泳分析呈现梯状外观。用其他测试鱼类病毒的DNA获得的阴性结果证实了该检测方法的特异性。BoHV-1-LAMP检测方法的分析灵敏度为1fg BoHV-1 DNA（稀释度为10(6)）。所开发的检测方法能够成功检测临床样本中的BoVH-1 DNA。本研究结果表明，所开发的BoHV-1-LAMP不仅是一种用于检测临床样本中BoHV-1的快速且高度灵敏的检测方法，而且还可用于区分野生型（gE阳性）和gE阴性BoHV-1病毒，这将改善埃及BoHV-1的防控计划。
