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

Feng Long; Yating Chen; Kaichuang Shi; Yanwen Yin; Shuping Feng; Hongbin Si

doi:10.3390/ani13040594

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

Animals (Basel). 2023 Feb 8;13(4):594. doi: 10.3390/ani13040594.

Authors

Feng Long¹, Yating Chen², Kaichuang Shi^{1

2}, Yanwen Yin¹, Shuping Feng¹, Hongbin Si²

Affiliations

¹ Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
² College of Animal Science and Technology, Guangxi University, Nanning 530005, China.

Abstract

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.

Keywords: NADC30-like PRRSV (NL-PRRSV); classical PRRSV (C-PRRSV); differential detection; highly pathogenic PRRSV (HP-PRRSV); multiplex Crystal digital RT-PCR (multiplex cdRT-PCR); multiplex real-time quantitative RT-PCR (multiplex qRT-PCR); porcine reproductive and respiratory syndrome virus (PRRSV).

Abstract

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