Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system

Abstract

The ability to visually detect low numbers of Salmonella in food samples is highly valuable but remains a challenge. Here we present a novel platform for ultrasensitive and visual detection of Salmonella Typhimurium by integrating the bio-barcode immunoassay (BCA), recombinase polymerase amplification (RPA), and CRISPR-Cas12a cleavage in a single reaction system (termed as BCA–RPA–Cas12a). In the system, the target bacteria were separated by immunomagnetic nanoparticles and labeled with numerous barcode AuNPs, which carry abundant bio-barcode DNA molecules to amplify the signal. Afterwards, the bio-barcode DNA molecules were amplified by RPA and subsequently triggered the cleavage activity of Cas12a to generate the fluorescence signal. Due to this triplex signal amplification, the BCA–RPA–Cas12a system can selectively detect Salmonella Typhimurium at the single-digit level with the naked eye under blue light within 60 min. Meanwhile, this novel platform was successfully applied to detect Salmonella Typhimurium in spiked milk samples with a similar sensitivity and satisfactory recovery, indicating its potential application in real samples. Furthermore, in virtue of the versatility of the antibody in the stage of BCA, the BCA–RPA–Cas12a system can be extended to further application in other bacteria detection and food safety monitoring.