IAFP 2009 Program Book and Abstract Book - AB138

Methods: Foods tested included hard salami, apple pie, tomato juice, nonfat dry milk, dry pet food, salad dressing, saw palmetto powder, soy flour, strawberry puree and dry cake mix. Soy flour and strawberry puree were naturally contaminated. Other foods were inoculated with yeast and/or mold (single strains or in combination) at target levels of 0, 20, 200, 2,000 and 10,000 CFU/g. Five replicate samples at each level were tested in parallel by the reference direct plating method and the Soleris method at four different test thresholds (10, 100, 1,000 and 5,000 CFU/g). Method agreement was calculated and results were compared using a Chi-square significance test. Inclusivity testing was conducted with 50 strains of yeast and mold (14 genera, 33 species) in pure culture, and all produced positive results. Exclusivity testing was performed with 30 strains of bacteria (17 genera, 29 species) in pure culture, and all produced negative results even when added to the Soleris vial at concentrations as high as 100,000 CFU/vial. Results: Agreement between the methods in individual trials ranged from 87 to 98% (89% overall). Chi-square analysis of results of individual trials showed, with few exceptions, that results of the Soleris and direct plating methods were not significantly different. Significance: Results showed that the Soleris method is an accurate alternative to conventional plating procedures for the semi-quantitative determination of yeast and mold in a variety of foods. The Soleris method offers considerable labor savings and provides results in 3 days or less, instead of the 5 days required by standard direct plating methods. P2-93 Isotachophoretic Method for the Concentration and Purification of Proteins and Nucleic Acids from Food Matrices Alex Proescher and CHARLES YOUNG JHUAPL, National Security and Technology, Laurel, MD, USA Introduction: It is envisioned that next generation biosensors will be capable of generating near real time results by use of multiple sensing modalities to yield orthogonal confirmation for the presence of pathogens in difficult matrices. For these systems, sample preparation will remain a critical challenge, requiring simultaneous co-purification of nucleic acids, proteins, lipids, and small molecules in a single, universal approach. Purpose: The purpose of this study was to evaluate the ability of an isotachophoretic approach for universal sample preparation by simultaneously concentrating and purifying proteins and nucleic acids from spiked food matrices for subsequent detection by immunoassays and real-time polymerase chain reaction assays (RT-PCR). Methods: A variety of food matrices and selective growth media were spiked with purified Bacillus atrophaeus DNA and ovalbumin and tested, using PCR and immunoassay respectively, to determine the effect of the matrix on the quality of the assay result. DNA and protein was added to food matrices or growth media previously determined to significantly alter the quality of the assay result and then purified and concentrated, using a gel-based isotachophoresis approach. The method utilized a “sea level” agarose gel format in which the buffers in the anode and cathode chambers of the gel box touched the gel, but were not allowed to mix. Application of a voltage resulted in the separation of sample components with purified nucleic acids and proteins located at the interface between a set of fast and slow moving electrolytes. Purified DNA and protein were removed from the gel matrix and the level of purification was evaluated based on characterization of the analytes by RT-PCR or immunoassays, depending on the target being measured. Results: For nine of the 11 matrices tested in this study, isotachophoresis purified target DNA to a point that the PCR result was indistinguishable from amplification of the target when no matrix was present. For the remaining 2 matrices, pepper and cocoa, a significant improvement in amplification efficiency after isotachophoresis was observed, but the amplification efficiency after purification was still poorer than amplification when no matrix was present. None of the matrices significantly affected the immunoassay result. Significance: This study provided a proof of concept, showing that an isotachophoretic approach to sample preparation could provide a solution for the simultaneous purification and concentration of both nucleic acids and proteins from food matrices. P2-94 An Independent Laboratory Evaluation of a Real-time PCR Combination Salmonella spp. Escherichia coli O157:H7 Method after a Common 8-hour Enrichment Compared to the USDA/FSIS Reference Methods for the Detection of Salmonella spp. and E. coli O157:H7 in Raw Ground Beef AMY C. REMES and Robert P Jechorek . rtech laboratories, St. Paul, MN, USA Introduction: The Salmonella spp./E. coli O157:H7 PCR method is intended to simultaneously reduce the enrichment time for Salmonella spp. from 20 h to 8 h and detect Salmonella spp. and E. coli O157:H7 from the same sample. This is accomplished using a single non-selective enrichment and DNA extraction protocol. Purpose: The purpose of this study was to compare the performance of the iQ-Check Salmonella spp. E. coli O157:H7 method to the USDA/FSIS (MLG 4.04 and MLG 5.04) reference methods for the detection of Salmonella spp. and E. coli O157:H7 in raw ground beef. Methods: In two separate trials, samples were simultaneously inoculated with Salmonella spp. and E. coli O157:H7. The test method samples were enriched for 8 h in buffered peptone water (BPW) and then the PCR assay was performed. The PCR results were compared to results of the 24 h enrichments for each reference method. Results: Statistical analysis of Chi Square according to Mantel-Haenszel showed no significant difference in the performance of the test method after an 8-h incubation compared to the 72-h and 46-h USDA/FSIS reference methods for the detection of Salmonella spp. and E. coli O157:H7. In the two trials, the test method identified 24/40 E. coli O157:H7 test samples, which were subsequently confirmed as positive, compared to results with the USDA reference method, which confirmed 19/40 samples. The test method identified 21/40 Salmonella spp. test samples, but only 7/40 of these 138 — Abstract Book Poster

IAFP 2009 Program Book and Abstract Book

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