Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens

Abstract

Background A diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field. Methods Three primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy. Results Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods. Conclusions The multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.

Development and application of a quadruplex real‐time PCR assay for differential detection of porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020

“…The detection limits of the conventional PCR assays specific for the four species of PCV strains were determined to be 2.8 × 10 2 copies/μl (Figure 3). The results indicated that the sensitivity of our newly developed quadruplex method is similar to previously reported multiplex real‐time PCR assays (Li et al, 2013; Li, et al, 2018; Zhang et al., 2020) but 10 times higher than the previously developed singleplex conventional methods (Tian et al., 2020; Yang et al., 2019; Zhang et al., 2020). To further evaluate the influence on the sensitivity when multiple pathogens were present, four representative PCV1‐4 positive samples were separately and simultaneously detected by the quadruplex real‐time PCR (Table S1).…”

“…PCV3 was first discovered in 2015 and reported to be associated with PDNS and abortion (Jiang et al, 2019;Palinski et al, 2017). PCV4 was recently detected from a severe diseased pig in China in 2019 (Zhang et al, 2020 (Li et al, , 2013;Tian et al, 2020;Yang et al, 2019;Zhang et al, 2020). However, all the developed methods Even though all four species of PCV strains have been detected…”

“…To further evaluate the quadruplex real‐time PCR assay, the same 120 clinical samples were detected by the new real‐time PCR assay and the conventional PCR assays. The singleplex conventional PCR assays were performed in the same reaction systems and amplification conditions as previously described (Tian et al., 2020; Yang et al., 2019; Zhang et al., 2020). The amplicons were analysed on 1.5% agarose gel electrophoresis in 1× TAE buffer.…”

“…Due to the newly identified PCV3 and PCV4 are difficult to isolate in vitro (Palinski et al., 2017; Zhang et al., 2020), molecular detection methods such as conventional and real‐time PCR assays are currently the major methods used for the detection of PCV infection. Several PCV differential detection methods (both conventional PCR and real‐time PCR) have been reported (Li et al, 2013; Li et al, 2018; Yang et al., 2019; Zhang et al., 2020). However, the assay suitable for simultaneously differential detection of four species of PCVs (PCV1, PCV2, PCV3 and PCV4) has not been established yet.…”

WITHDRAWN: Identification of Porcine circovirus type (PCV2) and type 3 (PCV3), Porcine 2 parvovirus (PPV) in swine by multiplex PCR test

“…However, those PCRs are designed for detection of a single viral agent at a time. Recently, duplex PCRs were developed for simultaneous detection of both PCV2 and PCV3 [26,27,28]. In the present study, we report the development and validation of a mPCR that allows simultaneous detection of these three important viruses in clinical samples.…”