Rapid Enzymatic\nDetection of Shiga-Toxin-Producing E. coli Using Fluorescence-Labeled Oligonucleotide\nSubstrates

Shiga-toxin-producing Escherichia coli (STEC) are important human pathogens causing diarrhea, hemorrhagic\ncolitis, and severe hemolytic uremic syndrome. Timely detection of\nthe multifaceted STEC is of high importance but is challenging and\nlabor-intensive. An easy-to-perform rapid test would be a tremendous\nadvance. Here, the major STEC virulence factor Shiga toxins (Stx),\nRNA-<i>N</i>-glycosidases targeting the sarcin ricin loop\n(SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based\nssDNA SRL substrates, which conferred a fluorescence signal after\ncleavage by Stx. Optimal results using bacterial culture supernatants\nor single colonies were achieved for substrate <b>StxSense 4</b> following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse\nSTEC serotypes, and <i>Shigella</i> were detected. Within\na proof-of-principle study, a total of 94 clinical strains were tested,\ncomprising 65 STEC, 11 <i>Shigella</i> strains, and 18 strains\nof other enteropathogenic bacteria without Stx. In conclusion, the\nassay offers rapid and facile STEC detection based on a real-time\nreadout for Stx activity. Therefore, it may improve STEC risk evaluation,\ntherapy decisions, outbreak, and source detection and simplify research\nfor antimicrobials.