Shiga-toxin-producing Escherichia coli (STEC) are important human pathogens causing diarrhea, hemorrhagic colitis, and severe hemolytic uremic syndrome. Timely detection of the multifaceted STEC is of high importance but is challenging and labor-intensive. An easy-to-perform rapid test would be a tremendous advance. Here, the major STEC virulence factor Shiga toxins (Stx), RNA-N-glycosidases targeting the sarcin ricin loop (SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based ssDNA SRL substrates, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. Within a proof-of-principle study, a total of 94 clinical strains were tested, comprising 65 STEC, 11 Shigella strains, and 18 strains of other enteropathogenic bacteria without Stx. In conclusion, the assay offers rapid and facile STEC detection based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection and simplify research for antimicrobials.
Keywords: FRET; N-glycosidase; Shiga toxin; Shiga-toxin-producing Escherichia coli detection; sarcin ricin loop; ssDNA SRL substrates.