Proof of concept for simultaneous and rapid detection of enterohemorrhagic eschericherichia coli (EHEC) serogroups by real-time reverse transcriptase PCR

Abstract

Foodborne illnesses caused by Enterohemorrhagic Escherichia coli (EHEC) are due to the consumption of contaminated food and water. Encoded by the eae gene, attachment and effacing lesions on human intestinal lining are produced by EHEC. The purpose of this study was to prove the concept that RNA from EHEC can be extracted and reverse transcribed to detect the eae gene. Previous studies detected eae in E. coli 0157 :H7 by real-time reverse transcriptase quantitative PCR (RT-qPCR); however, no studies have used RT-qPCR to detect eae in multiple EHEC strains. RNA from E. coli serogroups, 026, 045, 0 I 03, 0111, 0121, 0145, and 0157, was extracted with the Kingfisher Total RNA kit and reverse transcribed. Housekeeping gene, gstA, and virulence gene, eae, were detected with RT-qPCR. Crossing point (CP) values for gstA and eae with SYBR Green I qPCR averaged I 8. I 0 � 0.64 and 34.44 � 1.40, respectively. Hybprobe qPCR, used for eae comparison, resulted in lower CP values compared to SYBR Green, 22.70 � 1.46 versus 34.44� 1.40. Method specificity and detection I imit was assessed. Hybprobe qPCR was more specific and sensitive than SYBR Green l qPCR. The current study supports the proof of concept and hybprobe qPCR would be used as end-point the detection step in further experiments.