CRISPR/Cas Cell-Free Sensors for Rapid Detection of Pathogenic Escherichia Coli, Salmonella Enterica, and Listeria Monocytogenes in Complex Food and Environmental Samples

We created a detection system for pathogenic Escherichia coli, Salmonella enterica, and Listeria monocytogenes in complex sample matrices using a fluorescent cell-free assay called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). Foodborne illness costs Soldiers an average of 3 work days per year resulting in $900 million in lost productivity. We use magnetic beads coated with a fusion of mannose-binding lectin and antibody Fc region (FcMBL-MB) with affinity for numerous pathogenic bacteria for non-specific extraction/concentration to maximize signal. The system workflow includes five steps: incubation with FcMBL-MBs, magnetic separation and concentration, lysis, Recombinase Polymerase Amplification (RPA) and recognition by guide RNA to activate a CRISPR/Cas12 enzyme which cleaves ssDNA probes containing a fluorophore and quencher producing fluorescent signal commensurate with the target concentration. We identified target sequences for stx1 and stx2 for shiga toxin producing E. coli and invA for pathogenic Salmonella serovars to develop RPA primers and guide sequences. We tested thermally lysed dilutions of shiga toxin producing E. coli O157:H7 and non-target E. coli O127:H6 in a general test water standard. The LOD of E. coli O157:H7 was 1 CFU/mL (50 CFU total) for stx1 and 10 CFU/mL (500 CFU total) for stx2 assays. We investigated the effect of pH, incubation temperature, and sucrose concentration and found minimal effect on fluorescent signal. The results demonstrate great potential for customized molecular diagnostics in complex samples that are rapid, low SWaPC, and easy to use. The technology could be applied to molecular diagnostics for new biological threats with minimal redesign.