Asymmetric RPA-primed hybridization chain reaction enabling one-input-multiple-Cas12a activation for ultrasensitive...

Ensuring food safety requires accurate, simple detection of pathogens such as Salmonella. However, conventional methods suffer from long processing times, complicated procedures, and inadequate accuracy. To address this, we developed an integrated platform combining asymmetric recombinase polymerase amplification (aRPA), programmable hybridization chain reaction (HCR), and CRISPR/Cas12a readout in a simplified single-tube integrated format. Unlike conventional CRISPR assays, in which a single target activates a single Cas12a complex in a PAM-dependent manner, our design uses aRPA-generated single-stranded DNA to trigger HCR, converting each target into a long dsDNA molecule with multiple PAM sites. This architecture enables one-input, multiple-Cas12a activation, representing a shift from linear to cooperative amplification. Under optimized conditions, the assay achieves a detection limit of 1.8 × 10² colony-forming units/mL (CFU/mL) in direct screening, and reaches as low as 5 CFU/mL after a 6 h enrichment step. The method shows high specificity toward common foodborne pathogens and reliable performance in spiked food samples. Furthermore, a DNA releaser simplifies sample preparation and enhances convenience. In summary, this integrated strategy offers a reliable and practical tool for food safety monitoring.