Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China

Abstract

Porcine circoviruses (PCVs) are very small non-enveloped animal viruses with circular single-stranded DNA genomes of approximately 2 kb, which belong to the genus Circovirus in the family Circoviridae

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).…”

“…Conventional and real‐time PCR assays (both singleplex and multiplex) have been developed for the detection and differentiation of PCVs (Li et al, , 2013; Li, et al, 2018; Tian et al., 2020; Yang et al., 2019; Zhang et al., 2020). However, all the developed methods are targeting only one to three species of PCVs.…”

“…PCV4 was first detected in Hunan province in 2019 (Zhang et al., 2020). In addition, a latest study showed that PCV4 was also detected in Henan and Shanxi provinces (Tian et al., 2020). In this study, we first detected PCV4 in Jiangsu province, indicating that PCV4 is likely widespread in Chinese swine herds.…”

“…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.…”

“…To validate the PCR results, the ORF2 gene encoding the capsid protein was amplified from all PCV positive samples using primers described previously (Chen, et al, 2019; Chen, et al., 2019; Li et al, 2013; Tian et al., 2020). The amplicons were then submitted to Sanger sequencing (Genewiz, Suzhou, China).…”