Cloning of Porcine Circovirus 3 amplicon for molecular diagnosis purpose using TA cloning method

Abstract

Porcine circovirus 3 (PCV3), the third member of the genus Circovirus to infect swine, is a relatively newly identified virus associated with several health issues in pigs, including porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis, and multisystemic inflammation. Phylogenetic analyses indicate that PCV3 is globally prevalent, exhibiting significant genetic homogeneity across isolates (Klaumann et al., 2018). While the comprehensive impact of PCV3 on the swine industry is still under investigation, ongoing research focuses on its epidemiology, pathogenesis, immune response, and diagnostic methodologies. Polymerase chain reaction (PCR) is an essential technique in molecular biology and diagnostics, facilitating the extensive amplification of specific DNA sequences and enabling rapid, sensitive, and specific detection of pathogens. TA cloning is another pivotal technique that allows for the creation of positive controls by inserting known DNA fragments into a plasmid vector. In this study, a positive control for PCV3 was successfully constructed using both PCR and molecular cloning. A specific region of the PCV3 genome was amplified via PCR and subsequently cloned into a suitable vector. The resulting cloned fragment serves as a positive control for PCV3 detection assays, enabling precise validation and standardization of diagnostic tests. The application of PCR and cloning techniques in this context ensures the production of reliable and consistent positive controls for PCV3 research and diagnostics. The establishment of these positive controls is critical for improving diagnostic accuracy. These controls assist veterinarians and swine producers in making informed decisions regarding herd management, biosecurity measures, and outbreak response strategies. Additionally, these tools may also be adapted for similar research on other emerging swine pathogens, thereby expanding the impact of this study. Enhanced diagnostic capabilities from this research will facilitate the reduction of PCV3-related disease prevalence and its associated economic losses in the swine industry, thereby promoting overall economic stability.