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Indian J Virol. 2013 Jun;24(1):16-26. doi: 10.1007/s13337-012-0112-1. Epub 2012 Dec 16.

Development of a probe based real time PCR assay for detection of bovine herpes virus-1 in semen and other clinical samples.

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Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bangalore, 560 024 Karnataka India.
Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, 560 024 Karnataka India.
Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN 55108 USA.
Institute of Stem Cells, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, 560 065 Karnataka India.
Institute of Animal Health and Veterinary Biologicals, Bangalore, 560 024 India.


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 %.


Bovine herpes virus-1; Indirect ELISA; Real time PCR; Virus isolation

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