High-resolution melt curve PCR assay for specific detection of E. coli O157:H7 in beef. (April 2018)

• Record Type: Journal Article
• Authors: Liu, Yuejiao; Singh, Prashant; Mustapha, Azlin
• Is Part Of: Food control. Volume 86(2018)
• Page Start: 275
• Page End: 282
• Publication Date: 2018-04
• Subjects: E. coli O157:H7 -- Ground beef -- High resolution melt curve (HRM) PCR -- Immunomagnetic separation -- uidA; Food -- Quality -- Periodicals; Food -- Analysis -- Periodicals; Food handling -- Periodicals; Food industry and trade -- Quality control -- Periodicals; Aliments -- Industrie et commerce -- Qualité -- Contrôle -- Périodiques; Aliments -- Qualité -- Périodiques; Aliments -- Analyse -- Périodiques; Hygiène alimentaire -- Périodiques; Food -- Analysis; Food handling; Food -- Quality; Periodicals; Electronic journals; 664.07
• DOI: 10.1016/j.foodcont.2017.11.025
• Languages: English
• ISSNs: 0956-7135

Abstract

Abstract: Among the disease-causing Shiga toxin-producing Escherichia coli (STEC), E. coli O157:H7 is estimated to cause one-third of the total STEC illnesses and the most cases of hemolytic uremic syndrome (HUS) in the U.S. The uid A gene which is present in the majority of E. coli strains, codes for the synthesis of β-d -glucuronidase (GUD). In E. coli O157:H7, the uid A gene has a single point mutation at the +93 position that leads to an alteration in the amino acid sequence encoding the GUD enzyme. The aim of this study was to distinguish E. coli O157:H7 from other E. coli using a high resolution melt curve (HRM) real-time PCR assay. Based on the uid A mutation in E. coli O157:H7, a reliable PCR assay targeting the uid A gene was developed to differentiate E. coli O157:H7 from other E. coli serotypes and the closely related Shigella spp. The specificity of the assay was validated using a set of 128 pure bacterial DNA samples. Following enrichment and immunomagnetic separation (IMS), the assay was further applied to spiked ground beef and beef trim. Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily distinguishable from those of other E. coli serogroups and Shigella isolates in the PCR plot. Therefore, this assay was able to clearly discriminate E. coli O157:H7 strains from other E. coli and Shigella . With a 5–8 h enrichment time, followed by IMS, 10 CFU of E. coli O157:H7 were detectable in 325 g spiked beef samples. The HRM E. coli O157:H7 detection assay standardized in this study will allow for accurate identification of contaminated food samples and help in preventing foodborne outbreaks caused by this pathogen. Highlights: A high resolution melt curve PCR was developed for specifically detecting E. coli O157:H7. This assay avoided false negative and false positive results. A 5–8 h enrichment time allowed for detection of 10 CFU/g in 325 g beef. Immunomagnetic separation allowed for more specific capture of E. coli O157:H7. E. coli O157:H7 in beef was confirmed using Rainbow agar and serological testing.