PMC

Flow chart for concentration and RT-PCR electronic microarray detection of E. coli O157:H7 from spiked tap water and river water samples.

(A and B) Agarose gel electrophoresis analysis of the E. coli O157:H7 samples for the rfbE gene (A) and fliC gene (B). Lanes: 1, 100-bp DNA ladder; 2, RT-PCR product from sample containing 100 CFU; 3, DNase I control from 100 CFU; 4, RT-PCR product from 10 CFU; 5, DNase I control from 10 CFU; 6, RT-PCR product from 1 CFU; 7, DNase I control from 1 CFU; 8, RT negative control; 9, PCR negative control; 10, low-mass DNA marker. (C) Electronic microarray analysis of dilutions of E. coli O157:H7 cultures for the rfbE and fliC genes. Electronic microarray S/B ratios obtained from the E. coli O157:H7 rfbE and fliC targets amplified from 100, 10, and 1 CFU were positive. The S/B ratios from 100-C, 10-C, and 1-C (DNase I control samples) were negative. The error bars of S/B ratios represent one SD of the S/B ratio from triplicate samples.

(A) Induction of VBNC E. coli O157:H7 cells in deionized water at room temperature. Culturability was assessed by agar plate counting (in CFU per milliliter) (▪); the numbers of total cells (•) and of viable cells (▴) were determined by the Live/Dead method. Error bars represent SD. Downward-pointing arrows indicate culturability results below the level of detection. (B) Electronic microarray analysis of E. coli O157:H7 cells in a VBNC state spiked in 1 liter river water for the rfbE and fliC genes. The columns represent the fluorescence S/B ratios obtained with the electronic microarray chip. The S/B ratios were obtained from the E. coli O157:H7 rfbE and fliC targets amplified from the reverse transcription products obtained with 150 and 50 VBNC cells, and the ratios were positive; the S/B ratios obtained with the DNase I controls (150-C and 50-C) for each sample as described above (and with the river water control [River-C]) were negative. Error bars represent one SD of the S/B ratios from triplicate samples.