A comparison of methods for the detection of Escherichia coli O157 : H7 from artificially-contaminated dairy products using PCR

Rapid nucleic acid-based methods to detect human pathogens in foods are dep endent on the reliability of the DNA or RNA extraction method used. Skim mi lk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powde r were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food pr oduct using a solvent-based procedure and (ii) using a guanidinium isothioc yanate (GITC) procedure after previous bacterial concentration. Both the ef ficiency of DNA extraction and the overall PCR detection limits were evalua ted. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the p urity of the DNA obtained after bacterial concentration was significantly b etter than that obtained after organic extraction alone, PCR detection limi ts after each DNA recovery method varied with the specific food, ranging fr om 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yield s and subsequent PCR detection limits for reconstituted whey powder were ex tremely poor, and neither procedural changes nor the addition of PCR enhanc ement agents were able to improve recovery and/or detection. It is conclude d that the efficiency of DNA extraction is an extremely important and frequ ently overlooked variable impacting the overall detection limits of PCR-bas ed detection strategies.