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

Abstract 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 109 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.