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STANDARDIZATION OF REVERSE TRANSCRIPTION PCR FOR DETECTION OF ORBIVIRUS

KrishiKosh

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Title STANDARDIZATION OF REVERSE TRANSCRIPTION PCR FOR DETECTION OF ORBIVIRUS
 
Creator SATYASAMPARNA RAUT
 
Contributor Dr K. DHANALAKSHMI
 
Subject diseases, viruses, biological phenomena, livestock, pcr, rna, cell culture, sampling, elisa, electrophoresis
 
Description Orbivirus, the largest genus belonging to reoviridae family is apparently
responsible for emerging arboviral diseases of livestock. Among the diseases of livestock,
bluetongue is highly prevalent in India, particularly in south India. This present study was
undertaken for the purpose of standardising Reverse Transcription – Polymerase Chain
Reaction (RT-PCR) for detection of orbiviruses employing bluetongue virus spiked cell
culture, culicoides triturate and blood.
The current study targeted most conserved VP 1 segment coding RNA polymerase
gene for RT-PCR to amplify 188 bp length product for all orbiviruses. The protocol was
standardised for virus spiked cell culture media and culicoides triturate and a slightly
different protocol for virus spiked blood, successfully amplified the target length as
evident on agarose gel as clear single band at 188bp. The assay, after standardisation was
employed for detection in cell culture infected with different BTV serotypes 1, 2, 9, 10,
(xvi)
12, 16, 21, 23 and 24. All were positive with a specific 188bp product. The minimum
detection limit of virus was 1.0, 103 and 103 TCID50/ml for cell culture media, culicoides
triturate and blood respectively. A total of 28 blood samples from sheep and cattle with
pyrexia were tested for orbivirus but all were found negative. Possible reason could be no
infection or very low titre of virus.
The method standardised in the study can identify isolate to the level of orbivirus
genus. The application of this technique will unravel the circulation of other orbiviruses
hitherto not reported in the region. Use of this technology is anticipated to provide insight
into the appearance and distribution of known and unknown novel orbiviruses. The
current study thus provides the information to develop specific molecular diagnostic
assay that will allow confirmation of future outbreaks or cases of orbivirus infection
circulating along with BTV and retrospective analysis of previously unconfirmed cases,
hence facilitating epidemiological studies on these viruses and better control strategies
 
Date 2016-12-30T14:47:13Z
2016-12-30T14:47:13Z
2015-11-16
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/93734
 
Relation D;439
 
Format application/pdf
 
Publisher PVNR TVU