A single-tube RAA-CRISPR-Cas12a diagnostic platform for rapid detection of Chlamydia trachomatis

Chlamydia trachomatis, a pathogen of global public health importance, is prioritized by the World Health Organization as a major sexually transmitted infection. Its continued worldwide spread poses persistent challenges to reproductive health and disease control systems, underscoring the urgent need for rapid and accurate diagnostic methods. While current clinical diagnosis primarily relies on nucleic acid amplification tests (NAATs), their dependence on expensive thermal cyclers, complex laboratory setups, and sophisticated procedures limits widespread use in low-resource settings. Recent advances in recombinase-aided amplification (RAA) combined with CRISPR-Cas12a have revolutionized molecular diagnostics. This study established an integrated one-tube CRISPR fluorescence detection system by combining rapid nucleic acid lysis, isothermal amplification, and CRISPR-Cas12a cleavage. The system is coupled with a portable palm-sized 4-channel fluorescence detector and a terminal app for visual readout. Experimental results demonstrated a detection limit of 10¹ copies/mL with no cross-reactivity to common reproductive tract pathogens including Neisseria gonorrhoeae, Ureaplasma urealyticum, Candida albicans, Trichomonas vaginalis, and Gardnerella vaginalis. Clinical validation against qPCR showed 100.00% sensitivity (95% CI: 87.66–100.00%), 98.31% specificity (95% CI: 90.91–99.96), 98.85% overall agreement (95% CI: 93.76– 99.97), 96.55% positive predictive value (95% CI: 80.04–99.49), and 100.00% negative predictive value (95% CI: 93.84–100.00%).This successfully developed CRISPR-Cas12a-based rapid detection method offers simple operation and fast results – complete within 30 min using rapid lysis (5 min nucleic acid release, 20 min RAA amplification, and 5 min CRISPR cleavage) – making it suitable for primary care and field screening, thereby providing a novel strategy for early diagnosis of C. trachomatis infection.