Detection of viable Escherichia coli O157:H7 in food by propidium monoazide real-time polymerase chain reaction
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Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7. PMA is a dye that can penetrate dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Compared with ethidium monoazide (EMA), another DNA-binding dye, PMA has been reported to exert less influence on DNA amplification from viable cells. Three strains of E. coli O157:H7 (505B, G5310 and C7927) was prepared separately and serially diluted to generate cell suspensions ranging from 10 to 108 CFU/mL. Dead cells were obtained by heating the suspensions at 85oC for 15 min. Suspensions were then treated with PMA. The optimized assay was then applied to artificially contaminated apple juice and ground beef. DNA was extracted and amplified by TaqMan® real-time PCR targeting the uidA gene to detect only viable E. coli O157:H7 cells. Plasmid pUC19 was included in each reaction as an internal amplification control (IAC) to monitor the efficiency of real-time PCR. Results showed that a treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 108 CFU/mL dead E. coli O157:H7 cells. The optimized assay could detect viable E. coli O157:H7 at as low as 102 CFU/mL in pure culture, 104 CFU/mL in apple juice and 105 CFU/g in ground beef, in the presence of 106 CFU/mL or g dead cells. With 8 h enrichment, viable E. coli O157:H7 of 1 CFU/mL or g in apple juice or ground beef was detectable without interference from 106 CFU/mL or g dead cells. In conclusion, the PMA real-time PCR assay can effectively prevent amplification of DNA in dead cells of E. coli O157:H7 and differentiate viable E. coli O157:H7 from dead cells in apple juice and ground beef.