Multiplex real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3

Detection and phylogenetic analysis of porcine circovirus type 3 in central China

“…The clinical samples were strongly positive for PCV3, with Cq values between 14.9 and 39.08, and 35 tissue samples and 18 serum samples among the 170 clinical samples were found to be positive for PCV3. The overall positive detection rate was 31.18% (53/170, 95% CI: 23.92–38.43) (Table ), which was basically consistent with some previous reports (Stadejek et al., ; Kim et al., ; Ku et al., ), but lower than those reported by others (Zheng et al., ; Kwon et al., ). For 2013, 2014, 2015, 2016 and 2017, the PCV3‐positive detection rates were 23.08% (3/13, 95% CI: 3.67–49.83), 20% (1/5, 95% CI: 25.06–65.06), 42.86% (3/7, 95% CI: 0.95–86.66), 38.78% (19/49, 95% CI: 24.11–53.44) and 28.13% (27/96, 95% CI: 18.61–37.64), respectively.…”

“…In the assay, only PCV3 was detected to have a single melting peak, whereas no melting peaks were observed for the PCV2, PRRSV, PRV, PPV or ultrapure water. The detection limit of the standard plasmid was 2.19 × 10 1 gc/μl, which is comparable to that of previously reported qPCR assays (Chen et al., ; Kim et al., ; Wang, Zhang, Wang et al., ). The values of the intra‐assay standard deviation (SD) and the coefficient of variation (CV) ranged from 0.014 to 0.127 and 0.112% to 0.775%, respectively.…”

A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States

“…Using two non-overlapping targets for a given virus may not increase the assay's analytical sensitivity, but it will increase field strain coverage, and will help to detect strains with additional mutations, as the chance for a strain to have mutations on both target sites is small. Also, compared to the LOD of 50 copies per reaction in Kim et al (2017) and 90 copies per reaction in Zhang et al (2018), our assay appeared to be more sensitive: 17 copies per reaction of PCV3 and 14 copies per reaction of PCV2. The LOD of PCV2 was also evaluated with cell culture, with an LOD of around 1.4 TCID50 per reaction.…”

“…Considering the similar clinical symptoms caused by PCV2 and PCV3, and that PCV2 assays built many years ago are no longer covering the majority of field strains, there was a need to develop a mqPCR assay that was based on the most current sequencing data and capable of detecting the two viruses with high diagnostic coverage to field strains. Although several molecular detection methods were developed for rapid detection of PCV3 and PCV2 (Kim et al, 2017;Li et al, 2018;Zhang et al, 2018), our mqPCR assay showed apparent advantages. With the help of bioinformatics tools, 1907 PCV2 full genome sequences that were currently available in the GenBank were downloaded and analyzed to achieve high coverage in the assay design stage.…”

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3

“…Among numerous molecular diagnostic methods, real-time quantitative PCR (qPCR) enables the characterization of small quantities of sample with a high level of specificity, sensitivity, and accuracy. Several studies have used a qPCR assay for the detection of PCV3 [20,33,34] and PEDV [35,36], and the recently, a report described multiplex qPCR assay for the differential detection of PCV2 and PCV3 [37]; however, there are currently no reports validating the use of SYBR Green I -based duplex qPCR for the simultaneous detection of PEDV and PCV3 in the field. In this study, a SYBR Green I -based duplex qPCR assay was developed for the simultaneous detection of PEDV and PCV3, and its applicability was evaluated for the detection of PEDV and PCV3 in intestinal tissue and fecal samples of piglets suffering from diarrhea.…”