Loop-Mediated Isothermal Amplification for Rapid Detection and Differentiation of Wild-Type Bovine Herpesvirus-1 and Glycoprotein E-Deleted Marker Vaccine Strain

Abstract

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen affecting cattle and causing numerous reproductive disorders leading to significant economic losses to the cattle industry. The control programs for BoHV-1 are widely based on the use of glycoprotein E-deleted marker vaccines, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In this study, we report rapid and simple loop-mediated isothermal amplification (LAMP) assays for detection and differentiation of gE-deleted BoHV-1 from wild-type virus under isothermal conditions. The assays could be completed in 90 mintes, including viral DNA isolation, target amplification and visual interpretation of results with naked eye. The analytical sensitivity of the assays was 10 times higher than conventional PCR and could detect as little as 100 fg of viral DNA per reaction. The applicability of LAMP for detection of BoHV-1 in bovine semen was assessed by testing semen samples collected from breeding bulls and compared with TaqMan real-time PCR (as gold standard). The LAMP assays had diagnostic specificity of 100%. The diagnostic sensitivity was 88.2% and 83.3% for gB- and gE-LAMP, respectively, when compared with TaqMan real-time PCR. Our results have shown that the LAMP method developed in this study is a potential tool for rapid, sensitive, specific, cost-effective, and user-friendly detection and differentiation of wild type BoHV-1 from gE-deleted marker vaccine.