Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure

Yuan Chen; Shile Luo; Jianmei Tan; Luhua Zhang; Shengwu Qiu; Zhiyou Hao; Naidong Wang; Zhibang Deng; Aibing Wang; Qing Yang; Yi Yang; Changjian Wang; Yang Zhan

doi:10.3389/fmicb.2023.1092273

Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure

Front Microbiol. 2023 Feb 9:14:1092273. doi: 10.3389/fmicb.2023.1092273. eCollection 2023.

Authors

Yuan Chen¹, Shile Luo¹, Jianmei Tan¹, Luhua Zhang¹, Shengwu Qiu¹, Zhiyou Hao², Naidong Wang¹, Zhibang Deng¹, Aibing Wang¹, Qing Yang¹, Yi Yang¹, Changjian Wang³, Yang Zhan¹

Affiliations

¹ Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research Center of Reverse Vaccinology (RCRV), and Laboratory of Functional Proteomics (LFP), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, China.
² Animal Disease Prevention and Control Center of Yongzhou, Yongzhou, Hunan, China.
³ Animal Disease Prevention and Control Center of Hunan Province, Changsha, Hunan, China.

Abstract

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R ² values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.

Keywords: multiplex real-time PCR; porcine circovirus type 2; porcine circovirus type 3; porcine parvovirus; pseudorabies virus.