Detection and quantitation of Escherichia coli O157 in raw milk by direct qPCR

Abstract An improved method was developed to detect and quantify Escherichia coli O157 in raw milk to support studies on the food safety implications of consumption of raw milk. Centrifugation coupled with ethylene diamine tetra acetic acid (EDTA), sodium dodecyl sulfate (SDS), DNase and trypsin treatments under optimized conditions was used to concentrate bacteria from 10 mL of raw milk and remove polymerase chain reaction (PCR) inhibitors. DNA from the entire sample pellet was efficiently extracted in a fast, single-tube step, and quantitated with a 5′ nuclease quantitative PCR assay. This approach represents a significant improvement over earlier methods, in ability to use large sample volumes, effectively remove inhibitors without use of toxic or flammable reagents, efficiently extract DNA and obtain rapid, quantitative results. E. coli O157:H7 inoculated into raw milk was detected at 1 cfu mL−1 from a 10 mL sample in less than 3 h.