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

Yang Zhan; Lu-Hua Zhang; Yuan Lin; Yun-Feng Cai; Ya-Wen Zou; Zhi-You Hao; Zhan-Hua Luo; Nai-Dong Wang; Zhi-Bang Deng; Yi Yang; Chang-Jian Wang; Qing Yang

doi:10.1016/j.mcp.2021.101764

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

Mol Cell Probes. 2021 Oct:59:101764. doi: 10.1016/j.mcp.2021.101764. Epub 2021 Sep 15.

Authors

Yang Zhan¹, Lu-Hua Zhang², Yuan Lin³, Yun-Feng Cai², Ya-Wen Zou², Zhi-You Hao⁴, Zhan-Hua Luo⁵, Nai-Dong Wang⁶, Zhi-Bang Deng⁷, Yi Yang², Chang-Jian Wang⁸, Qing Yang⁹

Affiliations

¹ Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research Center of Reverse Vaccinology (RCRV), And Laboratory of Functional Proteomics (LFP), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Department of Veterinary Medicine, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Changde Research Center for Agricultural Biomacromolecule, Changde, Hunan, 415000, China. Electronic address: yangzhan@hunau.edu.cn.
² Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research Center of Reverse Vaccinology (RCRV), And Laboratory of Functional Proteomics (LFP), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Department of Veterinary Medicine, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China.
³ Department of Veterinary Medicine, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Animal Disease Prevention and Control Center of Hunan Province, Changsha, Hunan, 410128, China.
⁴ Yongzhou Animal Husbandry and Aquatic Affairs Center, Yongzhou, Hunan, 425000, China.
⁵ Dingcheng Animal Husbandry and Aquatic Affairs Center, Changde, Hunan, 415100, China.
⁶ Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research Center of Reverse Vaccinology (RCRV), And Laboratory of Functional Proteomics (LFP), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Department of Veterinary Medicine, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Changde Research Center for Agricultural Biomacromolecule, Changde, Hunan, 415000, China.
⁷ Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research Center of Reverse Vaccinology (RCRV), And Laboratory of Functional Proteomics (LFP), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China; Changde Research Center for Agricultural Biomacromolecule, Changde, Hunan, 415000, China.
⁸ Animal Disease Prevention and Control Center of Hunan Province, Changsha, Hunan, 410128, China.
⁹ Department of Veterinary Medicine, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, 410128, China. Electronic address: qingyanghn@hunau.edu.cn.

PMID: 34534618
DOI: 10.1016/j.mcp.2021.101764

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.

Keywords: African swine fever virus; B438L; B646L; Duplex; Real-time PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

African Swine Fever Virus* / genetics
African Swine Fever* / diagnosis
Animals
DNA, Viral
Genome, Viral / genetics
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Swine

Substances

DNA, Viral