Improved detection of Escherichia coli and coliform bacteria by multiplex PCR

Background: The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by beta-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential Escherichia coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific Escherichia coli detection are not easily identified. Results: In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and Escherichia coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. Conclusions: The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for Escherichia coli. Additionally, yaiO detection is non-redundant with enzymatic methods.