title: Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.
abstract: Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay  to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation,  at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation  of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7  isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E.  coli O157:H7 in raw milk and raw ground meat products.
authors.affiliation: Unité de Recherche CALITYSS/Equipe EMSA, VetAgro Sup, Université de Lyon, 1 avenue Claude Bourgelat, Marcy l'Etoile, France. s.miszczycha@vetagro-sup.fr
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Miszczycha
authors.forename: Sarah
authors.initials: S
authors.lastname: Ganet
authors.forename: Lysiane
authors.initials: L
authors.lastname: Duniere
authors.forename: Christine
authors.initials: C
authors.lastname: Rozand
authors.forename: Estelle
authors.initials: E
authors.lastname: Loukiadis
authors.forename: Delphine
authors.initials: D
authors.lastname: Thevenot-Sergentet
mesh_terms: D000818:Animals; D002417:Cattle; D002611:Cheese / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008461:Meat Products / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial