Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China

Pian Zhang; Zhaowen Ren; Xiaopeng Gao; Mengpo Zhao; Yanyun Wang; Jing Chen; Gang Wang; Hua Xiang; Rujian Cai; Shengjun Luo; Xiaohu Wang

doi:10.3389/fvets.2024.1353439

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China

Front Vet Sci. 2024 Apr 26:11:1353439. doi: 10.3389/fvets.2024.1353439. eCollection 2024.

Authors

Pian Zhang^#^{1

2}, Zhaowen Ren^#^{1

2}, Xiaopeng Gao¹, Mengpo Zhao^{1

2}, Yanyun Wang^{1

2}, Jing Chen¹, Gang Wang¹, Hua Xiang¹, Rujian Cai¹, Shengjun Luo¹, Xiaohu Wang¹

Affiliations

¹ Key Laboratory of Livestock Disease Prevention of Guangdong Province, Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Observation and Research Station for Animal Disease, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, China.
² College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.

^# Contributed equally.

Abstract

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R²) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Keywords: genotype; multiplex real-time PCR; phylogenetic analysis; porcine circoviruses; prevalence.

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the following grants: the Project of Collaborative Innovation Center of GDAAS (XT202207), the Scientific and Technological Plan Projects of Guangzhou (grant numbers 2023E04J1256 and 2023B04J0137), the Planning Funds for Science and Technology of Guangdong Province (grant numbers 2021B1212050021 and 2023A1111110001), Key Laboratory of Livestock Disease Prevention of Guangdong Project (2023B1212060040), Guangdong Provincial Forestry Department’s Provincial Financial Special Fund for Ecological Forestry Construction-Wildlife Conservation, the Guangdong Province Modern Agriculture Industry Technology System Pig Innovation Team Project.