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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
 
Creator Chandranaik, B.M.
 
Contributor Rathnamma, D.
Antony, P.X.
Shrikrishna Isloor
Veeregowda, B.M.
Renukaprasad, C.
Prabhudas, K.
Patil, S.S.
 
Subject ---
 
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.
 
Date 2016-07-22T14:53:37Z
2016-07-22T14:53:37Z
2012-05-08
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/69360
 
Language en
 
Format application/pdf
 
Publisher Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar