Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus. (October 2021) - Zhan, Yang
Abstract: An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/μL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health. Highlights: A developed real-time PCR assay detect two discontinuous ASFV genes. The duplex real-time PCR assay could prevent inaccurate results in one reaction. This method is specific for ASFV and showed high sensitivity. The assay has a best instrument suitability. A useful tool for controlling swine infection.