High-resolution Melt Curve PCR Assay for Detection of E. coli O157:H7 in Beef

Introduction: According to the CDC, 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 uidA gene which is present in the majority of E. coli strains, codes for the synthesis of the GUD enzyme. In E. coli O157:H7, the uidA gene has a single point mutation at the +93 position that leads to an alteration in the amino acid sequence encoding the GUD enzyme.

Purpose: The aim of this study is to distinguish E. coliO157:H7 from non-O157:H7 STEC serotypes using a high resolution melt curve (HRM) PCR assay.

Methods: Based on the uidA mutation in E. coli O157:H7, a reliable PCR assay targeting the uidA gene was developed to differentiate E. coli O157:H7 from other STECs and the closely related Shigella sp. The assay was validated using a set of 120 bacterial strains and spiked ground beef and beef trim.

Results: Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily distinguishable from those of non-O157 isolates in the PCR plot. Therefore, this assay was verified to be able to clearly discriminate E. coli O157 strains from other E. coli and Shigella. With a 6-h enrichment, 10 CFU E. coliO157:H7 were detectable in 325 g spiked beef samples.

Significance: Accurate detection of E. coli O157:H7 is critical in correctly determining the food source of this dangerous pathogen for preventing and controlling foodborne outbreaks.