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

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.