Development of real-time reverse transcription recombinase polymerase amplification (RPA) for rapid detection of peste des petits ruminants virus in clinical samples and its comparison with real-time PCR test

Abstract

Peste des petits ruminants (PPR), caused by small ruminant morbillivirus (SRMV), formerly called peste des petits ruminants virus (PPRV), is one of the most important pathogens in small ruminants, and has tremendous negative economic impact on the sheep industry worldwide. Current detection of PPRV in clinical samples mainly relies on real-time RT-PCR. Particularly, samples collected from rural area require highly equipped laboratories for screening. A rapid, real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA), employing primers and exo probe, was thus developed to perform at 42 °C for 20 min, and the detection limit at 95% probability was 14.98 copies per reaction and 0.326 TCID50/mL based on plasmid copy number and tissue culture infectivity titre. All the four lineages of PPRV could be detected with no cross-reaction to other pathogens including measles virus (MeV), goatpox virus (GTPV), canine distemper virus (CDV), foot-and-mouth disease virus (FMDV) and Mycoplasma capricolum subsp. capripneumoniae (Mccp). The performance of real-time RT-RPA assay was validated by testing 138 field samples and compared to real-time RT-PCR. The results indicated an excellent diagnostic agreement between real-time RT-RPA and a reference real-time RT-PCR method with the kappa value of 0.968. Compared to real-time RT-PCR, the sensitivity of real-time RT-RPA was 100%, while the specificity was 97.80%. The developed RT-RPA assay offers a promising platform for simple, rapid, and reliable detection of PPRV, especially in the resource-limited settings.