College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong, 030801, Shanxi, China.
China/WOAH Reference Laboratory for Classical Swine Fever, China Institute of Veterinary Drug Control, Beijing, China.
Virol J. 2023 Jul 14;20(1):150. doi: 10.1186/s12985-023-02111-1.
Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease.
Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains.
After optimization, the calibration curve showed good linearity (R > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 10 HAD/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 10, 1 × 10, and 1 × 10 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible.
The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.
非洲猪瘟（ASF）起源于非洲，于 2018 年传入中国。这种急性、高致病性传染病会影响家猪。世界动物卫生组织已将其列为法定报告疾病，中国也将其列为 A 类传染病。
针对四个 ASF 基因（B646L、EP402R、MGF505-3R 和 A137R）设计了引物和探针。与 21 株 ASF 毒株的基因序列相比，这些引物/探针具有高度保守性。
经过优化，校准曲线显示出良好的线性（R > 0.99），能够检测到的阳性质粒的最低浓度为 50 拷贝/μL，最低病毒载量检测限为 10 HAD/mL。此外，针对来自三种猪源 DNA 病毒的核酸和八种 RNA 病毒的 cDNA 进行四重实时荧光定量聚合酶链反应（qPCR）检测，均未出现扩增曲线，表明该方法具有特异性。此外，使用 1×10、1×10 和 1×10 拷贝/μL 的混合质粒进行四重 qPCR，组内三次重复测定的变异系数均<2%，表明该方法具有可重复性。
用不同基因缺失组合的 EP402R、MGF505-3R 和 A137R 株检测含可检测 EP402R、MGF505-3R 和 A137R 株的临床样本，结果符合预期。因此，该建立的四重 qPCR 方法可用于基因缺失型 ASF 病毒的分子诊断。
