College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Key Laboratory of Zoonotic Disease Prevention and Control of Guangdong, South China Agricultural University, Guangzhou, China.
Front Cell Infect Microbiol. 2024 Oct 23;14:1474676. doi: 10.3389/fcimb.2024.1474676. eCollection 2024.
Senecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease.
We designed multiple primer pairs targeting the most conserved region of the SVA gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA.
The SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×10 copies/µL, 8.76×10 ng/µL, and 1×10 TCID/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays.
The SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings.
塞尼卡病毒 A（SVA）是一种与仔猪急性死亡和猪水疱病相关的新型致病性病毒。SVA 的进一步流行将对全球养猪业造成相当大的经济损失。因此，快速准确的 SVA 诊断工具对于疾病的预防和控制至关重要。
我们设计了多个针对 SVA 基因最保守区域的引物对，并选择了特异性最高的引物对。随后，根据最高特异性引物对设计了 Nfo 探针。随后，完成了重组酶辅助扩增（RAA）反应，并优化了反应温度和时间。使用侧流设备（LFD）检测 RAA 扩增子。最后，建立了针对 SVA 的快速直观的 RAA-LFD 检测方法。
SVA RAA-LFD 检测方法可在 35°C 下反应 17 分钟内完成，结果可肉眼观察。然后，我们评估了该方法的性能。它表现出高度的特异性，与其他常见的猪病原体没有交叉反应。该方法对 pMD18-SVA-3D 质粒、DNA 扩增产物和病毒的最低检测限分别为 3.86×10 拷贝/μL、8.76×10 ng/μL 和 1×10 TCID/mL。然后使用 RAA-LFD、PCR 和 RT-qPCR 方法检测了 44 份临床样本。结果表明，RAA-LFD 与 RT-qPCR 检测方法的检测率一致。
本研究中建立的 SVA RAA-LFD 检测方法具有优异的特异性、灵敏度和省时特性，非常适合在缺乏仪器的实验室和现场环境中使用。
