A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus
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Date
2014
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Abstract
Background: Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as
sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and
ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A
SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection.
Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR
for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the
nucleocapsid (N) gene of PPRV.
Results: The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV
including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus
(NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of
33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture
infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV)
values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To
evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were
screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV
in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR.
Conclusions: The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific,
reproducible and rapid for detection and quantification of PPRV nucleic acids.
Description
TNV_BVR_2014_10(22)1-8
Keywords
Veterinary Science