Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction.
Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.
The University of Tennessee Food Safety Center of Excellence, Knoxville, Tennessee 37996, USA. soliver@utk.edu
L T
LT
Nguyen
B E
BE
Gillespie
H M
HM
Nam
S E
SE
Murinda
S P
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Oliver
D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008461:Meat Products / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D004269:DNA, Bacterial