An extraction-free one-step pathogen detection system (Ex-opods) based on LAMP and CRISPR-Cas12b

• Established rapid visual one-step LAMP-Cas12b detection system. • Co-integrated NTS sequence & mutant PAM into LAMP primers for activator amplification. • Screened sgRNA sequences for high efficiency, stability & universality. • Added HEX motif to LOOPF primer for one-pot amplification tracking. Isothermal amplification and gene editing-based in vitro diagnostics are highly sensitive and specific but can be challenging to design, develop, and use. To address this, we present a one-pot method that combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b for detecting Bordetella pertussis . This method incorporates a preferred sequence for sgRNA recognition and the mutant PAM recognized by Cas12b protein into the LAMP primers for B. pertussis , simplifying the design process. Our system can detect B. pertussis DNA at a concentration as low as 0.2 copies/μL. We also combined the technique with a nucleic acid-free extraction method, enabling extraction, amplification, and detection of B. pertussis from clinical specimens or cultures within one hour. We validated the system using 54 clinical samples of B. pertussis and found its sensitivity and specificity to be 100% and 92.90%, respectively, compared with Quantitative Real-time PCR (qPCR). This method offers greater design flexibility, is fast and sensitive, and holds promise for point-of-care testing for B. pertussis using the Ex-opods method.