Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus |
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Affiliation: | 2. Diagnostic Medicine/Pathobiology, Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States |
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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. |
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Keywords: | African swine fever virus Duplex Real-time PCR |
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