Development of a triplex crystal digital RT-PCR for the detection of PHEV, PRV, and CSFV

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV) are currently prevalent worldwide and cause similar neurological symptoms in infected pigs. It is very important to establish a detection method that can rapidly and accurately detect and differentiate these three viruses. Targeting the PHEV N gene, PRV gB gene, and CSFV 5' untranslated region (5'UTR), three pairs of specific primers and probes were designed, and a triplex crystal digital reverse transcription-PCR (cdRT-PCR) was developed to detect PHEV, PRV, and CSFV. The results indicated that this assay had high sensitivity, and the limitation of detection (LODs) for PHEV, PRV, and CSFV were 4.812, 4.047, and 5.243 copies/reaction, respectively, which was about 50 times higher than that of multiplex real-time quantitative RT-PCR (RT-qPCR). This assay showed good specificity, without cross-reaction with other important swine pathogens, i.e., FMDV, PRRSV, PEDV, SIV, TGEV, PoRV, and PCV2. This assay had high repeatability, with intra-assay coefficients of variation (CVs) of 0.73-1.87%, and inter-assay CVs of 0.57-2.95%. The developed assay was used to test 1,367 clinical tissue samples from Guangxi province in China, and the positive rates of PHEV, PRV, and CSFV were 3.44% (47/1,367), 1.24% (17/1,367), and 1.90% (26/1,367), respectively, with a coincidence rate of 98.98% and a Kappa value of 0.94 to the reference multiplex RT-qPCR. The established triplex cdRT-PCR was a highly rapid, sensitive, and accurate assay to detect and differentiate PHEV, PRV, and CSFV.