He Dalin, Zhao Saisai, Wang Fangfang, Wu Bingrong, Wei Feng, Zhao Yubo, Wei Xinhui, Ren Hui, Zhang Meijuan, Fan Yaru, Zhang Jiahao, Yu Shumin, Tang Yi, Diao Youxiang
College of Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, Shandong Province, China.
Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 10091, China.
Int J Biol Macromol. 2025 Apr;301:140474. doi: 10.1016/j.ijbiomac.2025.140474. Epub 2025 Jan 28.
H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10 copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.
H9N2禽流感病毒（AIV）是引起家禽呼吸道感染的主要病原体，对家禽业和人类健康构成重大威胁。早期检测和控制H9N2感染对于最大限度地减少经济损失和预防潜在的人畜共患病传播至关重要。一种名为CRISPR-Cas13a的新型CRISPR-Cas家族成员由CRISPR RNA（crRNA）和Cas13a核酸酶组成。通过基于crRNA对Cas13a进行重新编程，可获得一种特异性检测RNA的平台。在本研究中，我们开发了一种基于重组酶聚合酶扩增（RPA）的CRISPR-Cas13a诊断方法，用于快速检测H9N2 AIV。结果表明，通过荧光检测和侧流试纸条分别可在50分钟内检测到低至10拷贝/μL和10拷贝/μL的病毒，为H9N2检测提供了一种高灵敏度方法。该方法具有出色的特异性，可将H9N2与其他病原体区分开来。此外，基于RPA-Cas13a的检测系统在临床样本上进行了测试，表现出与逆转录定量聚合酶链反应（RT-qPCR）相当的性能。检测结果可通过侧流分析或荧光进行可视化，使其成为适用于现场、可在野外部署的诊断工具。总之，这种RPA-Cas13a诊断方法具有高可靠性、灵敏度和特异性，在临床和食品安全应用中快速检测H9N2及其他病毒病原体方面具有广阔的应用前景。
