Development of a Real-Time PCR for Escherichia coli based on gadE, an acid response regulatory gene

Increasingly, molecular methods have become important in identification and confirmation of bacteria at the species level. Rapid molecular methods provide sensitivity and specificity while reducing cost and resources. The primary goal of this study was to develop a real-time PCR assay for identification of Escherichia coli from an agar plate. GadE (gadE) directly regulates the glutamate-dependent acid response system (GDAR) in E. coli and is responsible for survival of at pH 2. Based on gene sequence data, a real-time PCR assay targeting gadE was developed for this purpose. Seventy bacterial isolates recovered from ground beef enrichments and 714 isolates from caecal contents were identified biochemically and tested with the real-time PCR assay developed in this study. The PCR assay and the biochemical identification had 100% agreement on the tested isolates. The gadE real-time PCR assay was demonstrated in this study to be an inexpensive, reliable method for confirming E. coli colonies within 1·5 h from an agar plate, thereby saving on final identification time. Significance and Impact of the Study There is always a need for rapid, inexpensive tests to identify bacterial cultures of public health concern at the species level. Recently, the numbers of tests performed to identify a culture as Escherichia coli have increased as regulations on pathogenic E. coli have increased as well as interest in the antibiotic resistance patterns of these organisms. The real-time PCR assay developed in this study provides a rapid, cost-effective way to identify E. coli from an agar plate without biochemical identification. This has importance for public health agencies in terms of analyst time and resource utilization.