title: Development of a probe based real time PCR assay for detection of bovine herpes virus-1 in semen and other clinical samples.
abstract: This study describes development of a TaqMan probe based real time PCR assay that can detect BoHV-1 of as low as 0.001 TCID50/0.1 ml in clinical samples, its comparative evaluation with indirect ELISA and virus isolation for detection of Bovine herpes virus-1 (BoHV-1) in semen and swab clinical samples. For this study, we collected samples from 212 animals (cattle and buffaloes) comprising 91 bulls and 121 females. Avidin-biotin ELISA employed on serum samples from 212 animals revealed 74 as seropositive for BoHV-1. On inoculation of semen/swabs on MDBK cell line, nine samples yielded cytopathic changes characteristic of herpes viruses. The isolates were confirmed by VNT and a conventional PCR. A real time PCR assay was standardised by designing a new set of TaqMan probe and primers targeting a 71 bp region on gB gene of the virus. The assay detected viral antigen in 21 seropositive and 14 seronegative animals, emphasizing the relevance of serology in BoHV-1 diagnosis, particularly in breeding stations. Further, real time PCR assay was 100 % sensitive and 87.19 % specific compared to virus isolation in detection of the BoHV-1 in clinical samples. The assay was validated at reputed national laboratories, with a sensitivity of ≥99 %.
authors.affiliation: Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: Basavegowdanadoddi Marinaik
authors.initials: BM
authors.lastname: Chandranaik
authors.affiliation: Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: Doddamane
authors.initials: D
authors.lastname: Rathnamma
authors.affiliation: Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: S S
authors.initials: SS
authors.lastname: Patil
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN 55108 USA.
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Kovi
authors.affiliation: Institute of Stem Cells, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, 560 065 Karnataka India.
authors.forename: Jyotsana
authors.initials: J
authors.lastname: Dhawan
authors.affiliation: Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: Shakunigowda
authors.initials: S
authors.lastname: Ranganatha
authors.affiliation: Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: Shrikrishna
authors.initials: S
authors.lastname: Isloor
authors.affiliation: Institute of Animal Health and Veterinary Biologicals, Bangalore, 560 024 India.
authors.forename: C
authors.initials: C
authors.lastname: Renukaprasad
authors.affiliation: Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, 560 024 Karnataka India.
authors.forename: K
authors.initials: K
authors.lastname: Prabhudas
keywords: Bovine herpes virus-1; Indirect ELISA; Real time PCR; Virus isolation