Detection of Five Shiga Toxin-producing Escherichia coli Genes with Multiplex PCR

Introduction: Escherichia coli serogroup O157 remains most commonly associated with foodborne outbreaks, but epidemiological studies suggest that non-O157 Shiga toxin-producing E. coli (STEC) causes 20-50% of STEC infections accounting for approximately 169,000 illnesses annually in the U.S. The ten most clinically relevant STECs belong to serogroups O26, O103, O111, O145, O157, O91, O113, O128, O45 and O121. Emerging strains such as the O104:H4 identified with the 2011 German outbreak could become more prevalent in the future.

Purpose: The purpose of this study is to develop a conventional multiplex PCR assay for the specific detection of STEC.

Methods: The multiplex PCR for STEC serotypes, O26, O45, O91, O103, O104, O111, O113, O121, O128, O145 and O157:H7 targets 5 different genes, stx1, stx2, eaeA, ehxA and uidA. To validate this PCR, 135 STECs, including one O157:H7 and three O157, were used for inclusivity (sensitivity) testing and 30 non-STECs were used for exclusivity (specificity) testing.  

Results: In 1.5 hours, from PCR analysis to results, all of the STEC DNA showed the presence of 1 to 5 amplification products, while the non-STEC DNA did not react to this multiplex PCR assay. In addition to being able to confirm O157:H7 isolates, it can identify non-O157:H7 STEC isolates that the current Bacteriological Analytical Manual O157:H7 multiplex PCR assay cannot detect. The uidA+93 SNP was retained in the assay to differentiate between O157:H7 and non-O157:H7 strains.

Significance: The rapid detection of STEC genes with the multiplex PCR assay will enable the rapid confirmation of virulent non-O157 STEC and O157:H7 food isolates, thus protecting the public health by enabling the identification and removal of these adulterated foods from the nation’s food supply.