One-step detection of Chlamydia psittaci using recombinase polymerase amplification and CRISPR/Cas12b

The development of rapid, sensitive, and user-friendly diagnostic methods for Chlamydia psittaci ( C. psittaci ) is critical for early diagnosis and effective control of infections. Current techniques, such as qPCR and next-generation sequencing, face limitations in accessibility and practicality for point-of-care testing (POCT). In this study, we present the WPTTS platform (one-step recombinase polymerase amplification (RPA)-CRISPR/Cas12b assay using w eak p rotospacer adjacent motif (PAM) recognition with t wo- t emperature s hifting), which leverages the temperature-dependent PAM stringency of Alicyclobacillus acidophilus Cas12b (AapCas12b) to overcome the challenges of one-step CRISPR detection. By employing weak PAM sequences and a two-temperature protocol (37 °C for amplification, 60 °C for detection), WPTTS minimizes interference between RPA amplification and CRISPR cleavage, achieving a sensitivity of 10 2 copies/reaction—equivalent to two-step methods—while maintaining operational simplicity. The platform also demonstrates enhanced specificity, enabling single-base resolution at critical target positions. Clinical validation with 40 samples confirmed its superior performance, with 95% positive agreement and 100% negative agreement compared to qPCR. This study highlights the potential of WPTTS as a robust, rapid, and highly sensitive tool for POCT applications in C. psittaci detection and broader infectious disease diagnostics.