Asymmetric RPA-primed hybridization chain reaction enabling one-input–multiple-Cas12a activation for ultrasensitive Salmonella detection

Apr 16, 2026Biosensors & bioelectronics

Sensitive Salmonella detection using a special DNA reaction that activates multiple Cas12a enzymes from one input

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Abstract

The assay achieves a detection limit of 1.8 × 10 colony-forming units/mL (CFU/mL) in direct screening.

The integrated platform combines asymmetric recombinase polymerase amplification, hybridization chain reaction, and CRISPR/Cas12a readout.
This design uses aRPA-generated single-stranded DNA to trigger cooperative amplification, allowing for multiple activations of Cas12a complexes.
The method demonstrates high specificity for common foodborne pathogens.
Performance is reliable in food samples spiked with pathogens.
A DNA releaser is included to simplify sample preparation.

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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 × 10 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. 2

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Asymmetric RPA-primed hybridization chain reaction enabling one-input–multiple-Cas12a activation for ultrasensitive Salmonella detection

Abstract

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