College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Department of Animal Health Standards and Regulation, China Animal Health and Epidemiology Center, Qingdao 266000, Shandong Province,China.
Virulence. 2024 Dec;15(1):2429022. doi: 10.1080/21505594.2024.2429022. Epub 2024 Nov 19.
Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and "collateral" promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT - qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.
猪流行性腹泻病毒（PEDV）是一种能引起猪流行性腹泻的病原微生物，它给养猪国家造成了巨大的经济损失。为了预防和控制 PEDV 感染，升级和快速的病毒检测技术是至关重要的。CRISPR-Cas13a 系统，即 CRISPR RNA（crRNA）的可编程性和 Cas13a 在识别靶 RNA 后的“旁系”混杂的核糖核酸酶活性，簇状规律间隔的短回文重复（CRISPR）-CRISPR 相关蛋白（Cas）13a 系统。在这项研究中，我们首次旨在开发一种基于重组酶聚合酶扩增（RPA）的 CRISPR-Cas13a 方法用于 PEDV 诊断。结果表明，在 37°C 下 40 分钟后，可检测到高达 10 拷贝/µL 的靶标 PEDV DNA 标准品。与其他选定的病原体相比，PEDV 检测表现出显著的特异性。此外，这种基于 RPA 的 CRISPR-Cas13a 方法可用于临床样本，与逆转录定量聚合酶链反应（RT - qPCR）的性能相似。使用侧向流动条或荧光可对我们提出的方法的结果进行可视化，从而便于在流行地区使用。总的来说，我们提出的方法表现出良好的可靠性、灵敏度和特异性，表明它可适用于检测其他病毒以诊断疾病和检查食品安全。
