Jan. 1, 2009 - Dec. 31, 2009Award Number
Carol D'lima, Ph.D.
University of California, Davis
Trevor Suslow, Ph.D.Resources
This project addresses key produce industry needs by validating novel methodologies and technologies to improve performance in specificity and sensitivity of pathogen testing across a diverse palette of commercial samples and environments. In order to obtain a broader understanding of the environmental biology and crop:pathogen interactions, methods are sorely needed to support comprehensive field investigations in a timely and efficient manner that protect grower confidentiality, business stability, and freedom from concerns for market exclusion based on findings. The evolving assessment of the appropriateness of current standards, critical limits, and “metrics” that were put into place as preventive measures for the safety of fresh produce requires that a detailed analysis of commercial plantings suspected of harboring contamination by human pathogens be conducted as often as allowable. The purpose of this project is to extend the public knowledge base that addresses verification of sampling methodologies and the detection and recovery platforms applied to pathogen testing programs for fresh produce. A cornerstone goal of this project will be to conduct a comprehensive analysis of available protocols and emerging technologies to stabilize and preserve pathogen detection enrichments and to have appropriate and verified protocols and tools to conduct strategic pathogen detection assays where commercially-initiated, pathogen-screening tests or relevant events (i.e. flooding, evidence of animal intrusion, and adjacent land activities) indicate a risk or concern. This sample “banking” is needed to more broadly secure grower participation and allow field access for detailed grid-analysis of crop and adjacent land, separated in time from crop marketing.
This project addresses industry-identified needs for effective human pathogen testing practices, focused on plant tissues but readily extended to preharvest inputs, environmental risk analysis, and postharvest applications. The specific objectives will first verify methodologies to stabilize and store primary enrichment samples for delayed analysis. This will be done by assessing DNA stabilization and extraction techniques using newly developed variations of the Whatman FTA card. These formats have the anticipated advantage of long term storage capacity without degradation of target DNA for pathogen detection. FTA cards will be tested with various inoculated plant and environmental samples over at least a nine month period. Detection from stored cards will compare commercially available rapid pathogen test kits and newly develop detection systems for E. coli O157:H7, EHEC, and Salmonella. For one set of methods development, paired plant samples will be inoculated on the leaf surface with low amounts (5, 50, 500, and 5000 CFU/leaf) of pathogenic E. coli and then allowed to dry. These plant samples would then be directly rubbed on a version of FTA cards termed Plant Saver. A second set of inoculated leaves, handled in the same manner, would be subjected to a quick homogenization step using a Bioreba ball-bearing tissue grinder), then applied to the 'FTA plant saver' card. In a further technique assessment, a small amount of non-selective enrichment buffer would be infused into an inoculated sample and held for 2, 4, and 6 hours under high relative humidity to allow for in situ amplification prior to direct application of 'enriched' leaves or homogenized samples. The purpose of these comparative evaluations of both homogenization and in situ enrichment is to expand our ability to conduct high density data point evaluations of contamination distribution and survival on individual leaf samples or small composites. This enabling industry and research tool will result in the development of a standardized, simple, decision-tree which would include an effective DNA extraction, specific to environmental and crop samples. To accomplish this, in part, a novel Real Time PCR detection technique will be improved and verified in on-farm sample backgrounds that will allow definitive screening for a broader spectrum of pathogenic E. coli. A TaqMan probe-based system will be developed with a view to diverse applications in assessment of the current 'metrics' testing system related to adjacent land use, fecal matter transference, soil amendments, and distribution of contamination within a lot. Furthermore, a probe based version of this primer set will be developed to ensure the specific detection of the presence of only pathogenic STEC and EHEC and once completed will be validated for use on environmentally contaminated samples. The final objective of the proposal would involve the necessary step of integrating and implementing these tools to carry out field evaluations on naturally contaminated samples. Results of these evaluations would test the actual capacity and capabilities of the developed tools to eliminate or resolve 'false positive' outcomes with other pathogen screens as well as provide science-based information regarding BMP's for preharvest pathogen testing on fresh produce.