Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat

Stéphane D Miszczycha , Sarah Ganet , Lysiane Duniere , Christine Rozand , Estelle Loukiadis (1) , Delphine Thevenot-Sergentet

1 LEM - Laboratoire d'Ecologie Microbienne - UMR 5557

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 z93 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 z191 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 41.5uC) 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.

Domaines: Sciences du Vivant [q-bio]

Stéphane D Miszczycha, Sarah Ganet, Lysiane Duniere, Christine Rozand, Estelle Loukiadis, et al.. Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat. Journal of Food Protection, 2012, 75 (8), pp.1373-1381. ⟨10.4315/0362-028x.jfp-11-498⟩. ⟨hal-04119442⟩