title: 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 24h 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.
authors.affiliation: U.S. Food and Drug Administration, San Francisco District Laboratory, 1431 Harbor Bay Parkway, Alameda, CA 94502, USA. andrew.lin@fda.hhs.gov
authors.forename: Andrew
authors.initials: A
authors.lastname: Lin
authors.forename: Omar
authors.initials: O
authors.lastname: Sultan
authors.forename: Henry K
authors.initials: HK
authors.lastname: Lau
authors.forename: Evelyn
authors.initials: E
authors.lastname: Wong
authors.forename: Gary
authors.initials: G
authors.lastname: Hartman
authors.forename: Carol R
authors.initials: CR
authors.lastname: Lauzon
mesh_terms: D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping; D022621:Shiga Toxin / Q000032:analysis / Q000096:biosynthesis; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification* / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity
chemical_list: D022621:Shiga Toxin