Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/µL for the multiplex qRT-PCR and 3.20 × 10-1 copies/µL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

Simultaneous detection of classical PRRSV, highly pathogenic PRRSV and NADC30-like PRRSV by TaqMan probe real-time PCR.

Porcine Reproductive and Respiratory Syndrome (PRRS), an acute infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is one of the most devastating diseases affecting the global swine industry. In order to establish a multiplex real-time PCR method for the simultaneous detection of the classical PRRSV (C-PRRSV) strain, the highly pathogenic PRRSV (HP-PRRSV) strain and NADC30-like PRRSV (NL-PRRSV) strain, we designed specific primers and TaqMan fluorescent probes based on the Nsp2 target gene sequence of these three different PRRSV strains, and designed American-type PRRSV (PRRSV-U) special primers and probes based on the relatively conserved target gene sequence of ORF7. The method established in this study can quickly and accurately detect and differentiate three types of strains of clinical tissue samples, respectively. This method plays a key role in the rapid diagnosis and determination of PRRSV.