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| Establishment and Evaluation of Fluorescence Quantitative PCR Method Based on the African swine fever virus MGF100-1L Gene |
| DONG Rui1, RUI Xian1, SHI Zheng-Wang2, PAN Yang-Yang1, BAO Shi-Jun1, ZENG Qiao-Ying1, ZHU Zi-Xiang1,2,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 State Key Laboratory of Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China |
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Abstract African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease with extremely high mortality. This study aimed to develop a specific and sensitive real-time PCR (qPCR) assay for detecting ASFV. The MGF100-1L gene (GenBank No. MK333180.1), belonging to the multigene families (MGFs) of ASFV, was analyzed to design and screen specific primers and probes targeting conserved regions. A recombinant plasmid containing the target gene fragment was constructed as a standard for optimizing the qPCR reaction system. The established ASFV qPCR method was evaluated for specificity, sensitivity, reproducibility, and agreement. A total of 90 clinical samples were tested and compared with the method recommended by the World Organisation for Animal Health (WOAH). Results showed that the standard curve linear equation was Y=-3.304X+38.793 with a correlation coefficient of 0.99, indicating good linearity. The limit of detection (LOD) was 0.87 copies/µL, demonstrating sensitivity comparable to the WOAH method. Specificity analysis confirmed no cross-reactivity with Classical swine fever virus (CSFV), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Porcine circovirus type 2 (PCV2), or Foot-and-mouth disease virus (FMDV). In clinical sample testing, the method showed high agreement with the WOAH method (Kappa=0.903, P<0.01) and significantly improved detection rates for weakly positive samples (4 additional samples detected compared to the WOAH method). This study successfully established a qPCR detection system based on the ASFV MGF100-1L gene, exhibiting excellent specificity, sensitivity, and high consistency with existing standard methods. This method provides a more sensitive and reliable technical tool for clinical ASFV detection, particularly for early diagnosis and control.
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Received: 12 June 2025
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Corresponding Authors:
* zhuzixiang@caas.cn
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