Application of Semi-quantitative M Gene-Based Hydrolysis Probe (TaqMan) Real-Time RT-PCR Assay for the Detection of Peste des petitis ruminants Virus in the Clinical Samples for Investigation into Clinical Prevalence of Disease

Abstract

Peste des petits ruminants (PPR) is a contagious, notifiable and economically important transboundary viral disease of small ruminants. In this study, a hydrolysis probe-based real-time reverse transcription-polymerase chain reaction (rt RT-PCR) assay for the detection and semi-quantification of PPR virus (PPRV) nucleic acid was developed using the virus RNA and matrix (M) gene-specific primers with Hex-labelled fluorescent probe and applied for the detection of PPRV in clinical samples to identify outbreaks and to monitor the prevalence of disease. The assay was found specific with a sensitivity detection limit of 0.5 pg of total PPRV RNA. Based on a serial dilution of the live-attenuated PPR vaccine virus, the detection limits were approximately 0.1 and 1 TCID₅₀ for the hydrolysis probe and conventional RT-PCR assays, respectively. The assay was linear within a range of 50 ng to 0.5 pg total virus RNA with an intra-assay coefficient of variation (CV) in the range of 0.91-2.86% and an inter-assay CV ranging between 0.59% and 2.37%. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the experimental/field clinical samples. This assay detected the PPRV in pre-clinical swab materials as early as the 4th day post-infection (dpi) and up to 17th dpi in nasal, ocular and oral swabs collected from experimentally infected animals. The rt RT-PCR was rapid, specific and 10 times more sensitive than conventional RT-PCR. It is an alternative test to the existing diagnostic assays and could be useful with enhanced applicability in field clinical diagnosis by avoiding the use of expensive commercial real-time PCR reagents. This assay was adopted directly in the detection of PPRV nucleic acid in clinical samples collected from sheep and goats suspected of PPR to monitor outbreak situations and the clinical prevalence of PPR in India.