O serogroup specific real time PCR assays for the detection and identification of nine clinically relevant non-O157 STECs

Abstract

TaqMan™ real time PCR assays were designed for each of the non-O157 STEC O serogroups most commonly associated with human illness: O26, O45, O91, O103, O111, O113, O121, O128, and O145. The nine RT-PCR assays can be run as single assays when a known pathogen is of concern, or multiplexed in three reactions, to quickly screen for the most clinically relevant O serogroups. All assays included an internal amplification control constructed from the green fluorescent protein gene as an indicator of PCR inhibition. Of 103 strains tested, the inclusive tests accurately identified the O serogroup for 101 strains. The exclusive tests for each assay yielded no false positives for over 120 Escherichia coli strains and 23 non- E. coli bacteria tested. Furthermore, the RT-PCR assays were tested by inoculating four food matrices, milk, apple juice, lettuce, and ground beef, at ≤30 CFU/25 g or mL. Following a 24 h selective enrichment, the RT-PCR assays detected STECs in all foods except for one ground beef sample inoculated with O111, and all apple juice samples inoculated with O113. The assays could also detect each O serogroup in human stool specimens inoculated with STECs at 1000 CFU/0.5 g of stool following 24 h enrichment.