title: EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains.
abstract: 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 (Tm) 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.
authors.affiliation: a Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India.
authors.forename: Sachin S
authors.identifier: http://orcid.org/0000-0002-4685-5106
authors.initials: SS
authors.lastname: Pawar
authors.affiliation: a Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India.
authors.forename: Chetan D
authors.initials: CD
authors.lastname: Meshram
authors.affiliation: b School of Animal Biotechnology , Guru Angad Dev Veterinary and Animal Sciences University , Ludhiana , India.
authors.forename: Niraj K
authors.initials: NK
authors.lastname: Singh
authors.affiliation: c Division of Biochemistry , Indian Veterinary Research Institute , Izatnagar , India.
authors.forename: Mohini
authors.initials: M
authors.lastname: Saini
authors.affiliation: a Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India.
authors.forename: B P
authors.initials: BP
authors.lastname: Mishra
authors.affiliation: a Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Gupta
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology; D006566:Herpesviridae Infections / Q000662:veterinary / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000145:classification / Q000235:genetics; D016014:Linear Models; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000145:classification / Q000235:genetics
chemical_list: D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins
keywords: Bovine herpesvirus-1; EvaGreen; differentiation; wild-type