Development of Real-Time PCR Methodology for the Enumeration of Low-Numbers of Salmonella and E. Coli in Ground Beef and Raw Vegetables without Enrichment

Non-Technical Summary:<br/>
There is a critical need in the meat processing and raw vegetable processing industries for the development of a rapid method for detection of infectious bacteria such as Salmonella and E. coli O157:H7 in such products well before shipping, so as to prevent infectious outbreaks and costly recalls.
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Approach:<br/>
Methodology will involve: (1) Homogenizing or stomaching 25 grams of ground beef (seeded with varying numbers of target bacteria) with at last 20 grams of beta-cyclodextrin in a total volume of 200 ml in saline to bind fat and exclude all bacteria. (2) Using differential centrifugation at 1,000g to sediments solids and 16,000g to pellet suspended bacterial cells. (3) Final pellet is then suspended in 30 ml of saline that is then mixed for 15 min with 11 grams of activated charcoal coated with bentonite in a 400 ml beaker to remove PCR inhibitors. (4) Bacterial cell suspension with charcoal is then passed through glass wool and resulting water clear filtrate is centrifuged at 16,000g to sediment bacterial cells. (5) Pellet is then made up to 1.0 ml with lysing solution and heated to boiling for 10 min. (6) Lysed cells are then centrifuged at 10,000g for 5 min to pellet debri and 10 microliters of DNA solution incorporated into PCR reactions using primers specific for the target organism. (7) Agarose gel electrophoresis will then be used to resolve the amplified band visualized under UV with "red safe" fluorescent dye, photographed and the fluorescent band quantified using the NIH 1.62 scanware program. Resulting rapid methodology will be be suitable for use by industry to prevent recalls. With leafy vegetables such as spinach and lettuce 250g of seeded eaves (held at 4 C overnight) will be vigorously rinsed with 200 ml of 0.025% SDS. Samples will then be subjected to differential centrifugation and steps 3 to 7 performed.