Sensitive and simultaneous detection of hygiene indicator bacteria using an enhanced CRISPR/Cas system in combination with a portable fluorescence detector

A rapid and sensitive diagnostic system is essential for the on-site monitoring of sanitary conditions in the food industry. In this study, we developed a point-of-care platform for the detection of hygiene indicator bacteria, including E. coli, coliform, and total bacteria, using a portable fluorescence detector. Through the dual recognition process by PCR and CRISPR/Cas, our system enables the simultaneous detection of bacteria in three different groups with the high specificity. The optimal reaction conditions obtained by the systematic investigation achieved a detection sensitivity as low as 10-1 cells/μL (1 cell/reaction). Furthermore, spiking tests in real samples were successfully carried out, which demonstrated the utility of our portable system for food hygiene monitoring. The proposed system represents a key platform for the sensitive and specific detection of hygiene indicator bacteria in resource- or facility-limited areas.