Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR

Abstract

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 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 100 to 108CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 108dead E. coli O157:H7cells/mL. The optimized assay could detect as low as 102CFU/mL viable E. coli O157:H7 in pure culture and 105CFU/g in ground beef, in the presence of 106/mL or g of dead cells. With an 8-h enrichment, 1CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 106dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells.