Abstract
BackgroundPeste 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.ResultsThe 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.ConclusionsThe 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.
Highlights
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 gastrointestinal tract leading to severe diarrhea [1]
We developed highly sensitive N gene target SYBR Green I real time Reverse transcription-polymerase chain reaction (RT-PCR) for specific detection and quantification of peste des petits ruminants virus (PPRV) in clinical samples
Summary
Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. 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. The causative agent of this disease, peste des petits ruminants virus (PPRV), is a member of the Morbillivirus genus in the Paramyxoviridae family. It has a negative sense single-stranded RNA genome encoding eight proteins [2]. The N gene of morbilliviruses is the most expressed gene due to a transcriptional gradient from the 3′ to the 5′ end of the genome and it is well-conserved gene [3] This gene is probably the best target for the development of a highly sensitive detection method
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