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R esearch
(Published online:
05-11-2015)
3. Development and evaluation of loop-mediated
isothermal amplification assay for rapid detection of
Capripoxvirus - Kanisht Batra, Aman Kumar, Vinay
Kumar, Trilok Nanda, Narender S. Maan and Sushila Maan
Veterinary World, 8(11): 1286-1292
doi:
10.14202/vetworld.2015.1286-1292
Kanisht Batra:
Department of Animal Biotechnology, College of Veterinary
Sciences, LLR University of Veterinary and Animal Sciences,
Hisar, Haryana, India;
drkanishtbatra@gmail.com
Aman Kumar:
Department of Animal Biotechnology, College of Veterinary
Sciences, LLR University of Veterinary and Animal Sciences,
Hisar, Haryana, India;
amankumar34237@gmail.com
Vinay Kumar:
Department of Animal Biotechnology, College of Veterinary
Sciences, LLR University of Veterinary and Animal Sciences,
Hisar, Haryana, India;
2008v60b@gmail.com
Trilok Nanda:
Department of Animal Biotechnology, College of Veterinary
Sciences, LLR University of Veterinary and Animal Sciences,
Hisar, Haryana, India;
nandatrilok@rediffmail.com
Narender S. Maan:
Resource Faculty, Department of Animal Biotechnology, College of
Veterinary Sciences, Lala Lajpat Rai University of Veterinary
and Animal Sciences, Hisar, Haryana, India;
narendermaan108@gmail.com
Sushila Maan: Department of Animal Biotechnology, College
of Veterinary Sciences, LLR University of Veterinary and Animal
Sciences, Hisar, Haryana, India;
sushilamaan105@gmail.com
Received: 20-07-2015, Revised:
19-09-2015, Accepted: 30-09-2015, Published online: 05-11-2015
Corresponding author:
Sushila Maan, e-mail: sushilamaan105@gmail.com
Citation:
Batra K,
Kumar A, Kumar V, Nanda T, Maan NS, Maan S (2015) Development
and evaluation of loop-mediated isothermal amplification assay
for rapid detection of
Capripoxvirus,
Veterinary World 8(11):
1286-1292.
Abstract
Aim:
The present
study was undertaken to develop a nucleic acid-based diagnostic
assay loop-mediated isothermal amplification assay (LAMP)
targeting highly conserved genomic regions of Capripoxvirus (CaPVs)
and its comparative evaluation with real-time polymerase chain
reaction (PCR).
Material and
Methods:
Lyophilized
vaccine strain of sheeppox virus (SPPV) was used for
optimization of LAMP assay. The LAMP assay was designed using
envelope immunogenic protein (P32) coding gene targeting highly
conserved genomic regions of CaPV responsible for causing sheep
pox, goat pox, and lumpy skin disease in sheep, goat and cattle
respectively. Serial tenfold dilution of SPPV recombinant
plasmid DNA was used for a calculating limit of detection.
Analytical sensitivity and specificity were performed.
Results:
The test
described is quick (30 min), sensitive and specific for
detection of CaPVs. The described assay did not show any
cross-reactivity to other related viruses that cause apparently
similar clinical signs. It was found to be ten times more
sensitive than conventional PCR however, 100 times less
sensitive than quantitative PCR (qPCR). LAMP assay results were
monitored by color change method using picogreen dye and agarose
gel electrophoresis.
Conclusion:
LAMP
assay can be a very good alternative for CaPV detection to other
molecular techniques requiring sophisticated equipments.
Keywords:
Capripoxvirus,
loop-mediated isothermal amplification assay, real-time
polymerase chain reaction, sensitivity,
specificity.
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