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 TCID50/0.1ml 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 71bp 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%.