An isothermal CRISPR-based diagnostic assay for Neisseria gonorrhoeae and Chlamydia trachomatis detection

作者：Hao Luo, Lihong Zeng, Xiaona Yin, Yuying Pan, Jianjiang Yang, Mingjing Liu, Xiaolin Qin, Zhanqin Feng, Wentao Chen, Heping Zheng · 发表于：Microbiology Spectrum · 年份：2023 · DOI：10.1128/spectrum.00464-23 · 被引用次数：11 · 研究领域：Insect symbiosis and bacterial influences、Syphilis Diagnosis and Treatment、HIV Research and Treatment

ABSTRACT The occurrence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) is increasing worldwide, particularly in low- and middle-income countries. In cases where most infections are asymptomatic and remain in a coinfection condition. It may cause missed detections or the severe subsequent symptoms. Accurate diagnosis and convenient screening are essential for disease control. However, molecular diagnostics for CT/NG predominantly rely on polymerase chain reaction (PCR)-based detection, which requires special devices and conditions. Here, we developed an isothermal CRISPR-based CT/NG dual-target detection system by pairing recombinase polymerase amplification (RPA) and CRISPR-Cas12a/13a detection. Multiplex RPA can amplify NG and CT simultaneously, and a single-pot reaction with the Cas12a/13a can be performed for further recognizing the amplified target, driving the cleavage of the corresponding fluorescence reporter separately; thus, the signal can be monitored in different channels. This CRISPR-based CT/NG detection system achieved analytical sensitivities of 10° copies/μL for both synthetic CT/NG dsDNA and exhibited no cross-reaction with other species. In tests on 88 clinical samples, our CRISPR-based assay showed excellent agreement with the commercial assay Roche Cobas 4800 (100% accuracy for NG, 94.32% accuracy for CT, and 97.73% accuracy for CT/NG coinfection) and showed higher positive percent agreement, negative percent agreement, and accuracy than thos...