Sample processing strategies for optimal PCR detection of pathogens in foods

by Isonhood, James Harold

The research in this manuscript highlights new and improved methods to concentrate pathogens from a complex food matrix and detect them via PCR. These contributions to the science of rapid pathogen detection are unique in that they are designed to measure the efficacy of the concentration or capture technique without using a pre-enrichment step. To address the potential of pathogen concentration to facilitate PCR detection, the following objectives were performed: (i) evaluating the performance of differential centrifugation as a means to concentrate and clarify the food sample for rapid PCR detection of Listeria monocytogenes, without pre-enrichment and (ii) investigating the efficacy of a novel immuno-capture device to capture E. coli O157:H7 and Salmonella typhimurium from foods.

For the first objective, we investigated filtration followed by a two-step, differential centrifugation as a means to concentrate bacteria and remove a large portion of the food sample prior to DNA extraction, PCR amplification, and Southern hybridization of L. monocytogenes targeting a unique region of 16S rDNA. Simple high speed centrifugation (11,950 x g) was also investigated to test the efficacy of our two-step method. Our method incorporated use of a 11g sample of ready-to-eat deli salad diluted 1:10 with 99ml 0.9% sterile normal saline. The two-step method was able to reduce the sample volume by approximately 10-fold rather than only 5-fold for simple high speed centrifugation. The two-step method was 1,000 fold (106 to 103 CFU/g) more sensitive than when using high speed centrifugation alone, and bacterial recoveries indicated that both methods produced similar recoveries. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 105 CFU/g for chicken salad, 104 CFU/g for macaroni salad, and 103 CFU/g for potato and seafood salads, with no pre-enrichment.

In our second objective, we evaluated the efficacy of a novel immuno-capture system (Pathatrix?) to capture S. typhimurium in buffered peptone water (BPW), ground turkey and nonfat dry milk and E. coli O157:H7 from BPW, ground beef and romaine lettuce. The Pathatrix system is unique in that it is designed to sample an entire 25g sample by circulating the homogenized 250ml volume across a surface of immunomagnetic beads. The samples were seeded with bacteria at levels ranging from 106 to 100 CFU/25g. The E. coli O157 format was able to capture 100 % of input E. coli O157:H7 and PCR amplification was able to detect the pathogen at 100 CFU/25g. The Salmonella format was not as robust, only capturing approximately < 1 % of cell input, but was able to produce a significant PCR detection limit of 102 CFU/25g when Pathatrix was preceded with the two-step centrifugation method. The two-step centrifugation further clarified the sample and improved detection by 1000-fold (105 to 102 CFU/25g).

This research provides further clues to expedite sample processing throughput prior to employing rapid methods for detection of pathogens in foods. It is our hope that knowledge of these techniques can help reduce or eliminate the need for preenrichment when screening food systems via PCR and other rapid methods.