Purpose: To optimize trace back and control of future outbreaks and to rapidly identify the presence of potentially virulent STEC, a PCR-based Luminex suspension array was developed to detect the genes coding for four virulence factors (stx1, stx2, eae, and ehxA) plus the O157:H7 specific +93 uidA single nucleotide polymorphism and an internal amplification control (IAC).
Methods: Multiplex PCR was performed in 25 μl reactions containing 1.2 pg IAC template using Qiagen® multiplex PCR plus kit under amplification conditions: 95°C for 15 min; 30 cycles, each cycle consisting of 95°C for 30 s, 60°C for 90 s and 72°C for 30 s; plus a final extension step at 68°C for 10 min. Reactions were analyzed in a Bio-Plex 200 instrument after oligonucleotide-microsphere conjugation and microsphere hybridization. Signal–to-background ratios were calculated from the Median Fluorescent Intensities (MFI) using the Bio-Plex Manager 6.0 software; ratios > 5.0 were considered as positive for each analyte.
Results: The inclusivity tests 100% accurately identified six stx2, 21 eae, six ehxA variant and the 45 STEC strains collected from various sources. An exclusivity panel consisting of 46 strains of non-STEC bacteria did not exhibit any false-positive signals. The Luminex suspension array identified STEC virulence genes in a 96-well plate format in less than 4 h. Addition of IAC excludes false-negative results due to PCR inhibitors in the reactions.
Significance: The rapid detection of these STEC genes will identify the presence of potentially virulent O157:H7 and non-O157 STEC. This assay can easily be expanded to include other STEC virulence genes or O serogroups of interest and is applicable to screening food enrichments.