A polymerase chain reaction (PCR)-mass spectroscopy assay was developed to identify non-O157 Shiga toxin-producing Escherichia coli (STEC) with Plex-ID biosensor system, a platform identifying short PCR amplicons by specific base compositions. This assay simultaneously amplifies five fragments of two housekeeping genes, two subunits of stx2 gene, and four other virulence genes of STEC. A total of 164 well-characterized STEC isolates were examined with the assay to build a DNA base composition database. Another panel of 108 diverse STEC isolates was tested with the established database to evaluate the assay's identification capability. Among the 108 isolates, the assay specificity was 100% for three (stx1, eae, and aggA) out of five tested virulence genes, but 99% for stx2 and 96% for hlyA, respectively. Main stx1/stx2 subtypes and multiple alleles of stx1/stx2 could be differentiated. The assay successfully identified several clinically significant serotypes, including O91:H14, O103:H25, O145:H28/NM, O113:H21, and O104:H4. Meanwhile, it was able to group isolates with different levels of pathogenic potential. The results suggest that this high-throughput method may be useful in clinical and regulatory laboratories for STEC identification, particularly strains with increased pathogenic potential.
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