Molecular Epidemiology of Bovine Herpes Virus -1 and Development of Real Time PCR Based Antigen Detection Kit for Diagnosis of Bovine Herpes Virus -1
KrishiKosh
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Title |
Molecular Epidemiology of Bovine Herpes Virus -1 and Development of Real Time PCR Based Antigen Detection Kit for Diagnosis of Bovine Herpes Virus -1
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Creator |
Chandranaik, B.M.
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Contributor |
Rathnamma, D.
Antony, P.X. Shrikrishna Isloor Veeregowda, B.M. Renukaprasad, C. Prabhudas, K. Patil, S.S. |
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Subject |
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Description |
Ph.D. Thesis
The present study describes the molecular epidemiology of Bovine herpes virus -1 (BoHV-1) and development of a Real Time PCR based antigen detection kit for diagnosis of BoHV-1 in clinical samples. The study included a total of 212 samples comprising semen from 91 bulls and swab samples from 121 cows. Blood samples were collected from all 212 animals. Avidin Biotin- ELISA employed on serum samples found 74 animals as seropositive for BoHV-1 antibodies. Upon inoculation of 212 samples on MDBK cells for virus isolation, samples from nine animals yielded cytopathic changes characteristic of herpes viruses. The isolates were confirmed by virus neutralisation test and a conventional PCR targeting gB gene of BoHV-1. The PCR amplicons of 443bp were cloned in pGEM T vector, nucleotide sequences were deduced and phylogenetic tree was constructed. Sequence analysis clustered five of these isolates under BoHV-1.1 subtype with highest sequence identities with Indian and European isolates of BoHV-1.1. The other four isolates were grouped as BoHV-1.2 with 100 % sequence identities with a European BoHV 1.2 isolate. A Real time PCR based detection kit was developed by designing TaqMan probe and primers targeting a 71bp conserved region on gB gene of BoHV-1. The developed assay was able to detect BoHV-1 upto 0.001TCID50 / 0.1ml. The assay detected BoHV-1 in twenty one seropositive and fourteen seronegative animals, emphasizing the relevance of serology in BoHV-1 diagnosis, particularly in breeding stations. The developed Real time PCR was 100 % sensitive and 87.19 % specific in detection of the BoHV-1 in clinical samples, when compared to virus isolation. The diagnostic kit was validated at National laboratories, with a validation sensitivity of ≥ 99 per cent. |
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Date |
2016-07-22T14:53:37Z
2016-07-22T14:53:37Z 2012-05-08 |
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Type |
Thesis
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Identifier |
http://krishikosh.egranth.ac.in/handle/1/69360
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Language |
en
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Format |
application/pdf
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Publisher |
Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
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