A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay

作者：Yunkai Zhang, Yan Xiang, Dengyong Hou, Liben Fang, Shuqi Cai, Jianping Zhang, Yujia Wang, Yuyu Jiang, Bin Liu, Jie Bai, Yue Ding, Jingjing Fang, Shuanghong Chen, Xingguang Liu, Xiaomeng Ren · 发表于：BMC Microbiology · 年份：2025 · DOI：10.1186/s12866-025-03882-z · 被引用次数：18 · 研究领域：Mosquito-borne diseases and control、CRISPR and Genetic Engineering、Insect symbiosis and bacterial influences

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 offe...