Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta

BACKGROUND: Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. RESULTS: Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 degrees C for 8min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15min incubation at 37 degrees C. The MIRA-LFD system achieved precise identification of the target species within 4min at 37 degrees C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 x 10(-1)ng mu L-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. CONCLUSION: Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30min and 10min, respectively), at 37 degrees C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. (c) 2024 Society of Chemical Industry.