Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains

Shengnan Ruan; Wenhui Ren; Bin Yu; Xuexiang Yu; Hao Wu; Wentao Li; Yunbo Jiang; Qigai He

doi:10.3390/v15091946

Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains

Viruses. 2023 Sep 18;15(9):1946. doi: 10.3390/v15091946.

Authors

Shengnan Ruan^{1

2}, Wenhui Ren^{1

2}, Bin Yu^{1

2}, Xuexiang Yu^{1

2}, Hao Wu^{1

2}, Wentao Li^{1

2}, Yunbo Jiang^{1

2}, Qigai He^{1

2}

Affiliations

¹ National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
² The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes.

Methods: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay.

Results: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/μL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes.

Conclusion: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.

Keywords: PRRSV; abortion; diagnosis; genotyping; quadruple; respiratory distress.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
China / epidemiology
Female
Porcine Reproductive and Respiratory Syndrome* / diagnosis
Porcine respiratory and reproductive syndrome virus* / genetics
Pregnancy
Reverse Transcriptase Polymerase Chain Reaction* / veterinary
Sus scrofa
Swine

Grants and funding

This work was supported by the China Agriculture Research System of MOF and MARA (CARS-35), the comprehensive prevention and controlling of important diseases in intensive pig farms and construction and demonstration of disease-free compartment. (2021BBA085), and Yingzi Tech & Huazhong Agricultural University Intelligent Research Institute of Food Health (No. IRIFH202209).