4.6 Article

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

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MICROBIOLOGY SPECTRUM
卷 -, 期 -, 页码 -

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AMER SOC MICROBIOLOGY
DOI: 10.1128/spectrum.00464-23

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Neisseria gonorrhoeae; Chlamydia trachomatis; coinfection; CRISPR-Cas12a; CRISPR-Cas13a; dual-target detection

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A CRISPR-based isothermal CT/NG dual-target detection system was developed, which demonstrated high sensitivity, excellent agreement with commercial assays, and greater convenience compared to PCR-based methods.
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 degrees copies/mu L 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 those reported for TaqMan PCR in a previous study. In addition, the results of this CRISPR-based system could be acquired within 75 min. Our isothermal diagnostic method with promising performance provides more convenience than PCR-based methods for detection and screening.

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