The Detection Of Salmonella Based On CRISPR-Cas12a System

Foodborne pathogens are present in a variety of foods and can enter the food supply chain at all stages of production,processing,distribution and marketing.Salmonella is one of the main causes of foodborne pathogens,so the detection of Salmonella in food is necessary to reduce the incidence of foodborne diseases.In recent years,the existing laboratory detection methods have been very mature,but these methods have their own shortcomings,such as time-consuming,inconvenient to use.Therefore,there is an urgent need to develop a rapid,highly sensitive and specific field detection method of pathogenic bacteria.Two methods for Salmonella detection based on CRISPR-Cas12a platform were developed in this paper.One is G-quadruplex-based CRISPR-Cas12a bioassay for ultrasensitive and highly specific detection of pathogenic bacteria.Compared to the previous bacteria detection strategies,G-quadruplex-based CRISPR-Cas12a bioassay not only takes full advantage of the high sensitivity and specificity of CRISPR-Cas12a based diagnosis but also transforms the target recognition into visualized signal readouts to broaden the application.Integrating the G-quadruplex DNAzyme based colorimetric reaction,the detection results could be directly visualized and distinguished by naked eyes as well as a smartphone with a Color Picker App.The performance of our proposed bioassay was outstanding and is complete within 3 h.The lowest detection limit was 1CFU/m L,the detection range was 10~0 to 10~8 CFU/m L,and there was a good linear relationship(R~2=0.993).The method is easy to operate and avoids the dependence on complicated instruments and special detectors,and has been successfully applied to the detection of Salmonella in various food samples,such as beer,juice.The one is a combination of PMA dye and CRISPR-Cas12a system for the detection of viable pathogens.The method not only takes advantage of the high sensitivity and specificity of CRISPR-Cas12a system,but also combines a novel nucleic acid dye(PMA)and sodium lauryl sarcosinate(sarkosyl).It can effectively inhibit the amplification of genomic DNA in dead bacteria and eliminate the signal interference of dead bacteria during detection.The combination of PMA dye and Cas12a system can quantitatively detect 0.1%of viable bacteria,which is suitable for Salmonella in beer and juice.The platform based on CRISPR-Cas12a system for Salmonella detection has high sensitivity,specificity and on-site detection capability.This not only expands the application scope of the CRISPR-Cas biosensor technology,but also can be used as an effective tool for food safety assessment.