Development and Validation of an RT-Droplet Digital PCR Assay for Detection of Peste des Petits Ruminants

To achieve rapid, accurate, and quantitative detection of peste des petits ruminants virus (PPRV), we aligned the available PPRV genomic sequences, screened for conserved regions, and designed specific primers and probes accordingly. An RT-droplet digital PCR (RT-ddPCR) method was established, and a series of reaction conditions were optimized. Subsequently, the specificity, sensitivity, and repeatability of the method were subsequently evaluated. The results demonstrated that the RT-ddPCR method exhibited good specificity, with positive results obtained exclusively for PPRV and negative results for all other goat-derived viral or bacterial biological products and seed viruses tested. The method showed promising analytical sensitivity, with a LOD of 1.0 copies/μL for gene copy number. The method also demonstrated good repeatability, with coefficients of variation in all groups not exceeding 5%. In a preliminary clinical evaluation using 150 field samples, RT-ddPCR identified 113 positive cases, compared to 105 by RT-qPCR, with 8 additional low-copy-number positives detected exclusively by RT-ddPCR, and although the sample size is limited for precise diagnostic performance estimation, the assay showed 100% relative diagnostic sensitivity (105/105) and 82.2% relative diagnostic specificity (37/45), with almost perfect agreement between the two methods (κ = 0.87). As the positive signals in these discordant samples were too weak for independent confirmation by Sanger sequencing, they should be interpreted as "presumptive positives" requiring further investigation. The RT-ddPCR method established in this study enables sensitive and rapid detection of PPRV nucleic acid. However, detection of low-copy-number targets should be interpreted with caution, as it does not necessarily indicate active infection or epidemiologically significant virus circulation. This assay shows promise as a valuable research and surveillance tool for PPRV nucleic acid detection, particularly for confirming low-level viral RNA in samples near the detection limit of conventional methods, and could complement existing RT-qPCR-based approaches in PPR eradication programs.