Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus

• Published in: Molecular and cellular probes Vol. 59; p. 101764
• Main Authors: Zhan, Yang, Zhang, Lu-Hua, Lin, Yuan, Cai, Yun-Feng, Zou, Ya-Wen, Hao, Zhi-You, Luo, Zhan-Hua, Wang, Nai-Dong, Deng, Zhi-Bang, Yang, Yi, Wang, Chang-Jian, Yang, Qing
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2021
• Subjects: African Swine Fever - diagnosis; African swine fever virus; African Swine Fever Virus - genetics; Animals; B438L; B646L; DNA, Viral; Duplex; Genome, Viral - genetics; Real-time PCR; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Swine; African swine fever virus; Real-time PCR; B646L; Duplex; B438L
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2021.101764

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. •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.