Salmonella is a type of common foodborne pathogen of global concern, seriously endangering human health. In molecular biological detection of Salmonella, the method of amplifying DNA often faces the problem of aerosol pollution. In this study, a microfluidic chip was developed to integrate loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to detect Salmonella. The LAMP reaction solution was initially injected into the chamber to amplify at 65 °C for 20 min; the CRISPR/Cas12a reaction solution was subsequently injected to mix with the amplicons for fluorescent signal production at 43 °C for 30 min. Then, the results can be confirmed by naked eyes under 495 nm light or by a fluorescence immunochromatographic reader. The detection limit of this method for Salmonella DNA was 118 pg/μL. The sensitivity and specificity of this method was 100%. Furthermore, this method was used to detect Salmonella after enrichment for 4 h in salmon and chicken samples spiked with 30 CFU/25 g, and was verified to have a stable detection capability in real samples. The microfluidic chip integrated with the LAMP and CRISPR/Cas12a system not only provides a possibility of highly sensitive endpoint fluorescent visual detection of a foodborne pathogen, but also greatly eliminates the risk of aerosol contamination.
Keywords: CRISPR/Cas12a; LAMP; Salmonella; visual detection.