Electronic deoxyribonucleic acid (DNA) microarray detection of viable pathogenic Escherichia coli, Vibrio cholerae, and Salmonella typhi

Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

“…Our studies and those of others demonstrate that the rfbE gene is a good viability marker for E. coli O157:H7 (23,41). The rfbE gene-based reverse transcription-PCR (RT-PCR) method reported by Yaron and Matthews (41) has a detection limit of 10 6 CFU, which does not provide sufficient sensitivity for monitoring culturable and/or VBNC cells of E. coli O157:H7 in water.…”

“…The principle of electronic microarray detection has been described in detail elsewhere (23). Briefly, three major steps are involved in this technique: immobilization of captures to individual spots on the chip; hybridization with targets; and detection of reporter fluorescence.…”

“…The signal-to-background (S/B) ratio represents the average of the fluorescence intensity values from three spots loaded with the same capture and target set relative to the background value. The threshold for a positive identification was an S/B ratio higher than 5 (23).…”

“…Similarly, a 100% conserved fragment in all 28 available sequences was identified and used to design the capture and reporter sets for the fliC gene. The capture and reporter sets for the rfbE and fliC genes were tested to specifically detect E. coli O157:H7 and not other bacteria, as described in our previous study (23). The electronic microarray analyses were also performed according to procedures that were previously described (23).…”

“…Using mRNAs encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC) of E. coli O157:H7 as the markers, we have developed a combined RT-PCR and electronic microarray technique for specific detection of viable E. coli O157:H7 (23). Microarray-based bacterial detection with 16S rRNA, various functional genes, or whole genomic DNA have been reported for a wide range of bacteria (5, 10, 11, 13, 34, 38-40, 42, 44, 45) because of the high-throughput nature of the microarrays.…”