Abstract

 Management of contaminated sediments has predominately focused on chemical contaminates, overlooking risks posed by pathogenic biological contaminants. Current analytical methods do not provide defensible data with respect to the presence of pathogens in sediment and their ability to cause disease in human and ecological receptors. Unfortunately, classical microbial methods that require enrichment, isolation and growth of microbial pathogens on nutrient media fail to detect most environmentally conditioned and dormant pathogens. Emerging molecular methods circumvent the problem of culturing potential pathogens by directly detecting the pathogen's DNA. In an effort towards adopting/adapting these new molecular technologies for assessing the risk posed by pathogens in sediments, we evaluated the sensitivity of a commercially available RT-PCR based TaqMan E. coli 0157:H7 amplification/detection kit. The sensitivity of this kit was empirically determined using sediment samples that were spiked with known amounts of E. coli 0157:H7 DNA. For comparison, parallel experiments were conducted looking at the sensitivity of the assay in pure water spiked with known amounts of E. coli 0157:H7 DNA. Our results indicate that the TaqMan assay could detect as little as 50 theoretical colony forming unites (CFUs) when pure water was spiked with known amounts of DNA. In contrast, in the presence of sediment, the detection limit of the assay was 200 theoretical CFUs. This 4-fold reduction in sensitivity can be attributed to the matrix effect of the sediment. Currently additional experiments are being conducted to determine the upper limit of sediment sample size that can be processed. A quantitative understanding of matrix effects on assay performance is necessary because pathogens, unlike their indicators, are present in only small quantities in the environments. To compensate for this more, the sample must be processed which in turn can negatively impact assay sensitivity.