Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of Porcine Circovirus 2, 3, and 4 in East China from 2020 to 2022

SIMPLE SUMMARY: Porcine circovirus (PCV) is widespread in the world and causes huge economic losses to the pig industry. Recently, the infection rates of PCV2 and PCV3 have been increasing year by year in China, and a new type of virus, PCV4, discovered in 2019, was found to result in complex pathogenic infection. In this study, a TaqMan-probe-based multiplex real-time PCR (qPCR) assay was developed for the differential detection of PCV2, -3, and -4. A total of 535 clinical samples from Shandong, Zhejiang, Jiangsu, and Anhui provinces, East China, were included in the analyses to evaluate the application of multiplex qPCR. The resulting data showed that the individual positive rates of PCV2, PCV3, and PCV4 were 35.33%, 40.37%, and 33.08%, respectively; the mixed infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 31.03%, 30.09%, and 30.84%, respectively; the mixed infection rate of the three viruses was 28.22%. The developed assay showed extreme specificity, high sensitivity, and excellent reproducibility for the simultaneous detection and differentiation of three porcine circovirus types. ABSTRACT: Porcine circovirus disease (PCVD) caused by porcine circovirus (PCV) is an important swine disease that is characterized by porcine dermatitis, nephropathy syndrome, and reproductive disorders in sows. However, disease caused by PCV2, PCV3, or PCV4 is hard to distinguish, so a rapid and sensitive detection method is urgently needed to differentiate these three types. In this study, four pairs of specific primers and the corresponding probes for PCV 2, -3, and -4, and porcine endogenous gene β-Actin as the positive internal reference index, were designed to establish a TaqMan multiplex real-time PCR (qPCR) assay for the simultaneous differential diagnosis of different types of viruses. The results showed that this assay has good specificity and no cross-reactivity with other important porcine viral pathogens. Furthermore, it has high sensitivity, with a detection limit of 101 copies/μL, and good reproducibility, with intra- and inter-group coefficients of variation below 2%. Subsequently, 535 clinical samples of suspected sow reproductive disorders collected from Shandong, Zhejiang, Anhui, and Jiangsu provinces from 2020 to 2022 were analyzed using the established assay. The results showed that the individual positive rates of PCV2, PCV3, and PCV4 were 31.03%, 30.09%, and 30.84%, respectively; the mixed infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 31.03%, 30.09%, and 30.84%, respectively; the mixed infection rate of PCV2, PCV3, and PCV4 was 28.22%. This indicated that this assay provides a convenient tool for the rapid detection and differentiation of PCV2, PCV3, and PCV4 in pig farms in East China. Our findings highlight that there are different types of porcine circovirus infection in pig farms in East China, which makes pig disease prevention and control difficult.