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Nucleic Acid And Antibody Based Detection Of Classical Swine Fever Virus

KrishiKosh

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Title Nucleic Acid And Antibody Based Detection Of Classical Swine Fever Virus
 
Creator Rathnapraba, S.
 
Contributor Kumanan, K.
Gunaseelan, L.
Saravanabava, K.
Vijayarani, K.
 
Subject Classical swine fever virus
5’UTR
E2
NS5B gene
RT-PCR
Monoclonal antibodies
Lateral flow test
LFT(Ag)
LFT(Ab)
 
Description Classical swine fever (CSF) is a highly contagious, fatal disease of
domestic pigs. The infection causes major economic losses in countries with
industrialised pig production. The present study was undertaken to isolate and
characterize classical swine fever virus (CSFV) from field outbreaks, to
develop nucleic acid and antibody based rapid diagnostic assays for detection
of CSFV. Field samples (n=114) collected from 15 swine fever suspected
outbreaks were screened by indirect FAT and reverse transcriptase PCR (RTPCR)
targeting three CSF viral genome specific regions, NS5B, E2 and 5’UTR.
The percent positivity by indirect FAT was 48%. The diagnostic sensitivity
(DSn) and diagnostic specificity (DSp) of FAT with NS5B PCR were
estimated as 89 % and 75% with an accuracy of 82%. The DSn and DSp of
FAT with E2 PCR were to be 90% and 78% with an accuracy of 84%. On
comparison with indirect FAT and three RT-PCR, 5’UTR gene specific
RT- PCR resulted in higher diagnostic sensitivity and specificity of 93% and
78% respectively with an accuracy of 85%. In this study period, six classical
swine fever isolates viruses were isolated and characterized by molecular
techniques. All these isolates were titrated by IFAVN and had a TCID50 titre
ranged from 2.3-2.6 at 5th passage and from 3.25 - 3.68 at 10th passage level in
PK15 cells. .
Genotypic analysis based on 5’UTR, E2 and NS5B genes of the field
isolates revealed circulation of 1.1, 1.2 and 2.1 genotypes in these outbreaks.
The CSFV-TS2 isolate (CSFV/AP/TRP2/2009) of 1.1 genotype was selected
for the production of monoclonal antibodies. From two fusion trials, a total of
143 clones were produced with a mean fusion efficiency of 20.2 % and 28%
positivity in indirect ELISA. Two positive hybridomas 3B10 and 2G6 were
subcloned twice by limiting dilution. In Western blotting, cell culture
supernatants of these were bound to 55 kDa protein of CSFV, suggestive of E2
protein and isotype as IgG2a, IgG2b respectively. The 3B10 Mab was further
purified by protein G affinity column and conjugated with colloidal gold.
Lateral flow test (LFT) for CSFV antigen detection was developed. The
LFT antigen (LFT-Ag) detection strip was used to screen 58 samples
(in-house) and 34 samples at College of Veterinary and Animal Sciences,
Assam. The results showed 45% and 53% positivity respectively and were
compared with standard tests. On comparison of LFT-Ag with RT-PCR (inhouse),
the sensitivity and specificity was 67% and 84% respectively. From the
34 samples screened at COVAS, Assam and on comparison with Prionics Ag-
ELISA, the percent positivity was 66% and 53% in Ag-ELISA and LFT
respectively. The detection sensitivity (DSn) and specificity (DSp) was 62%
and 64 % respectively. The antibody detection LFT (LFT-Ab) was developed
for detection of antibodies to CSFV. A total of 129 sera samples tested, of
which 44 samples were positive in indirect ELISA and 49 in LFT –Ab test with
a positivity of 34% and 38% respectively. The detection sensitivity and
specificity obtained was 86.4% and 87.1% respectively. Out of 65 samples
screened at Assam, 35 samples were positive with 58% positivity in LFT and
44 samples were positive with 55% positivity in Prionics ELISA kit for
antibody detection.
 
Date 2016-05-23T16:10:28Z
2016-05-23T16:10:28Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66235
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University