title: 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 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. 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 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells.
authors.affiliation: Food Science Program, Division of Food Systems and Bioengineering, University of Missouri, Columbia, United States.
authors.forename: Yarui
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Food Science Program, Division of Food Systems and Bioengineering, University of Missouri, Columbia, United States. Electronic address: MustaphaA@missouri.edu.
authors.forename: Azlin
authors.initials: A
authors.lastname: Mustapha
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival; D004269:DNA, Bacterial / Q000378:metabolism; D019453:Escherichia coli O157 / Q000235:genetics*; D005516:Food Microbiology / Q000379:methods*; D008027:Light; D057230:Limit of Detection; D008460:Meat / Q000382:microbiology*; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Ground beef; Propidium monoazide; Real-time PCR; Viable Escherichia coli O157:H7