Development of a SYBR Green-based RT-qPCR assay for the rapid detection of peste-des-petits-ruminants virus.

Abstract

Peste-des-petits-ruminants (PPR) is primarily a disease of small ruminants caused by peste-des-petits-ruminants virus (PPRV; Paramyxoviridae, Morbillivirus caprinae), formerly the small ruminant morbillivirus. PPRV can cause significant morbidity and mortality in small ruminants and a significant economic impact. Conventional reverse-transcription PCR (RT-PCR), and probe-based and SYBR Green-based RT quantitative real-time PCR (RT-qPCR), are employed for the molecular detection of PPRV. Here we describe a SYBR Green-based RT-qPCR for rapid and sensitive detection of PPRV. We designed the specific primers from the conserved region of the fusion gene (F) of PPRV. The standard curve of the established RT-qPCR assay had a good linear relationship. The developed assay was also 3 log units more sensitive than the conventional RT-PCR, with a detection limit of 13.6 copies and an efficiency of 98.2%. There was no cross-reactivity with other caprine respiratory viruses, namely bluetongue virus, goatpox virus, and orf virus. The positive detection rate of clinical samples was 11 of 64 (17.2%) versus 10 of 64 (15.6%) by conventional RT-PCR. We confirmed our results by sequencing the full F and N genes of the isolates. Our SYBR Green RT-qPCR can be used as a fast, economical, and sensitive alternative to RT-PCR for the detection of PPRV.