A CRISPR/dCas9-enabled, on-site, visual, and bimodal biosensing strategy for ultrasensitive and self-validating detection of foodborne pathogenic bacteria

It is imperative to develop practicable pathogenic bacteria detection methods. We devised a biosensor for the ultrasensitive detection of Salmonella typhimurium ( S. typhi ), termed as SCOUT-dCas9 (ultrasensitive, cross-validating, on-site, and dUal-mode test using CRISPR/dCas9). Simply, the species-specific invA gene of S. typhi was amplified using loop-mediated isothermal amplification with a biotinylated primer, which can be specifically “pulled down” by “antibody-like” dCas9-single guide RNA to form a ternary complex. SYBR Green I and streptavidin-modified alkaline phosphatase were used to functionalize them to generate fluorescent and colorimetric signals, respectively. With this strategy, the target could be dexterously converted into bimodal signals that were cross-validated to afford more reliable results. For both modes, SCOUT-dCas9 was able to detect as low as 1 CFU/mL with a dynamic range from 1 to 10 9 CFU/mL. Lastly, SCOUT-dCas9 had satisfactory selectivity and was capable of detecting S. typhi -contaminated real food samples. SCOUT-dCas9 provides a robust platform for bacterial detection.