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.
United States Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA, USA.
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Tillman
United States Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA, USA.
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Simmons
United States Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA, USA.
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JL
Wasilenko
United States Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA, USA.
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Narang
United States Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA, USA.
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Cray
United States Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, MD, USA.
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Bodeis-Jones
United States Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, MD, USA.
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G
Martin
United States Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, MD, USA.
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S
Gaines
United States Department of Agriculture, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Athens, GA, USA.
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Seal
D000818:Animals; D002417:Cattle; D002432:Cecum / Q000382:microbiology; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005798:Genes, Bacterial; D008460:Meat / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*
D029968:Escherichia coli Proteins; C479562:GadE protein, E coli; D014157:Transcription Factors
E. coli; Non-O157 STEC; acid resistance; gadE; real-time PCR