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P1-17 Detection of Escherichia coli O157H:7 and Non-O157 Shiga Toxin-producing E. coli by Triplex Real-time PCR

Monday, August 4, 2014
Exhibit Hall D (Indiana Convention Center)
Weimin Wang, U.S. Food and Drug Administration, Laurel, MD
Huanli Liu, U.S. Food and Drug Administration, Laurel, MD
Christopher Elkins, U.S. Food and Drug Administration, Laurel, MD
Baoguang Li, U.S. Food and Drug Administration, Laurel, MD
Introduction: Foodborne outbreaks caused by Escherichia coli O157H:7 and Non-O157 Shiga toxin-producing E. coli (STEC) remain an important food safety concern and a serious public health problem in the United States and worldwide.

Purpose: In order to effectively mitigate future outbreaks caused by this food-borne pathogen, it is necessary to have an ample availability of sensitive, specific, and reliable methodologies to detect E. coli O157:H7 and non-O157 STEC strains.

Methods: A triplex real-time PCR was developed for specific detection of E. coli O157:H7 by simultaneously targeting a putative fimbriae protein ORF Z3276 and its Shiga toxin (Stx1 and stx2) genes.

Results: The sensitivity of the triplex assay on the target genes of Z3276, stx1, and stx2 was found to be only slightly lower than that of those genes obtained with uniplex PCR assays. The detection limit for this triplex real-time PCR are 100 fg DNA, which is equivalent to less than 20 CFU per reaction. In inclusivity and exclusivity tests containing 182 bacterial strains, all E. coli O157:H7 (n = 133) were identified as positive, other strains including non-O157 (n = 40), Salmonella enterica (n = 3), and Shigella strains (n = 6) were not detected.

Significance: This triplex real-time PCR assay is sensitive and specific, and is useful in detection of E. coli O157:H7 and non-O157 STEC.

Back to: Poster Session - Applied Laboratory Methods, Produce, Low-water Activity, Beverages and Water
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