Zhang Yunkai, Xiang Yan, Hou Dengyong, Fang Liben, Cai Shuqi, Zhang Jianping, Wang Yujia, Jiang Yuyu, Liu Bin, Bai Jie, Ding Yue, Fang Jingjing, Chen Shuanghong, Liu Xingguang, Ren Xiaomeng
Naval Medical Center, Naval Medical University, 880 Xiangyin Road, Shanghai, 200433, China.
National Key Laboratory of Immunity & Inflammation, Naval Medical University, Shanghai, 200433, China.
BMC Microbiol. 2025 Mar 25;25(1):163. doi: 10.1186/s12866-025-03882-z.
Dengue Virus (DENV) is a life-threatening pathogen leading to dengue fever, which brings about huge public health challenges globally. However, traditional detection methods currently fail to meet the increasing demands of clinic practice in terms of speed, simplicity, and accuracy. To address these limitations, we developed a novel, rapid, and highly sensitive diagnostic method for universal DENV detection by integrating recombinase polymerase amplification (RPA) assay and the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system into one-pot. This approach achieves exceptional sensitivity and specificity for DENV detection, with the entire process completed within 40 min, without the need for sophisticated equipment. The limit of detection (LOD) was determined to be 91.7 copies/test. Using this one-pot RT-RPA CRISPR/Cas12a detection system, all four serotypes of DENV (1 to 4) were successfully identified. In terms of specificity, the assay accurately detected DENV-infected positive samples without cross-reactivity with four other interfering viruses-infected samples (VSV, SeV, HSV-1 and IAV). Furthermore, we established a universal DENV RT-RPA-CRISPR/Cas12a-lateral flow dipstick (LFD) platform, which successfully identified all four serotypes of DENV with a sensitivity of approximately 250 copies/test. Collectively, our method not only provides a robust alternative for universal DENV detection but also offers valuable insights for the identification of other viruses.
登革病毒（DENV）是一种危及生命的病原体，可导致登革热，在全球范围内带来巨大的公共卫生挑战。然而，传统的检测方法目前在速度、简便性和准确性方面无法满足临床实践日益增长的需求。为了解决这些局限性，我们通过将重组酶聚合酶扩增（RPA）检测与成簇规律间隔短回文重复序列（CRISPR）及相关的Cas12a蛋白（CRISPR/Cas12a）系统整合到一个反应体系中，开发了一种用于通用型DENV检测的新型、快速且高度灵敏的诊断方法。这种方法在DENV检测中实现了卓越的灵敏度和特异性，整个过程在40分钟内完成，无需复杂设备。检测限（LOD）确定为91.7拷贝/测试。使用这种一体化的逆转录RPA CRISPR/Cas12a检测系统，成功鉴定了DENV的所有四种血清型（1至4型）。在特异性方面，该检测方法准确检测出DENV感染的阳性样本，与其他四种干扰病毒（VSV、SeV、HSV-1和IAV）感染的样本无交叉反应。此外，我们建立了一个通用型DENV逆转录RPA-CRISPR/Cas12a-侧向流动试纸条（LFD）平台，该平台成功鉴定了DENV的所有四种血清型，灵敏度约为250拷贝/测试。总体而言，我们的方法不仅为通用型DENV检测提供了一种强大的替代方案，还为其他病毒的鉴定提供了有价值的见解。
