College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.
Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.
Front Cell Infect Microbiol. 2021 Mar 17;11:633763. doi: 10.3389/fcimb.2021.633763. eCollection 2021.
African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10-15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32-64-fold for direct PCR, while only a 2-4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.
非洲猪瘟（ASF）是一种高传染性且通常致命的猪传染病，被世界动物卫生组织（OIE）列为应报告疾病。自 2018 年中国首次爆发以来，它已在全球范围内造成巨大的经济损失。由于迄今为止尚无有效的疫苗可用，因此对 ASF 的诊断对于其监测和控制至关重要，尤其是在资源有限且环境简陋的远离城市的地区。在这项研究中，建立了一种敏感，特异，快速且简单的血液样本中非洲猪瘟病毒（ASFV）基因的分子即时检测方法，包括用简单稀释和煮沸 5 分钟处理血液样本，在 37°C 水浴中进行重组酶辅助扩增（RAA）10 分钟，以及在室温下通过侧向流动测定（LFA）进行目视读取，耗时 10-15 分钟。无需从血液样本中提取病毒 DNA，从采样到最终诊断决策的完整工作流程可以在 30 分钟内用最小的设备要求完成。RAA-LFA 对合成基因质粒的检测限为 10 拷贝/μl，比 OIE 推荐的 PCR 和实时定量 PCR 灵敏 10 倍。此外，在测试经典猪瘟病毒，猪繁殖与呼吸综合征病毒，猪流行性腹泻病毒，伪狂犬病病毒和猪圆环病毒 2 时，未观察到 RAA-LFA 的阳性读数，表现出良好的特异性。RAA-LFA 对临床血液样本的评估显示与 OIE 推荐的 PCR 具有 100％的吻合率，无论是在提取的 DNA 还是处理的血液样本中均如此。我们还发现血液样本中的某些成分极大地抑制了 PCR 的性能，但对 RAA 的影响很小。直接 PCR 时，血液至少稀释 32-64 倍可消除抑制作用，而直接 RAA 时仅适合血液稀释 2-4 倍，这表明当血液用作检测样本时，RAA 是比 PCR 更好的选择。综上所述，我们建立了一种敏感，特异，快速且简单的 RAA-LFA 用于 ASFV 分子检测，无需提取病毒 DNA，为将来在现场即时检测 ASF 诊断提供了良好的选择。
