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PCR is one of the most powerful analytical techniques ever developed. It allows segments of minute amounts of double stranded DNA to be amplified several million-fold in 2 to 3 hr. Its most notable application to foods is for the detection of food borne pathogenic and toxigenic bacteria in a wide variety of food products in addition to confirming the identification of such organisms isolated from foods. However, one of the limitations in the application of the PCR for detection and enumeration of food borne disease organisms is the presence of PCR inhibitors in complex food matrices that usually makes it impossible to detect low numbers (1 to 100 per gram) of target bacteria in foods. <P>Experience has shown that the various DNA purification resins, kits, and mini-spin columns available are not effective in completely removing such PCR inhibitors. This has resulted in the use of enrichment procedures that require 18 to 48 hrs. incubation. Such enrichments procedures and the incubation times involved negate the rapid time advantage of the PCR.<P> This investigator has recently developed the use of activated charcoal for the complete elimination of soluble PCR inhibitors from complex food matrices containing very high levels of potent PCR inhibitors, resulting in the detection of as few as 5 target organisms per gram of food without enrichment culture. <P>Our methodology involves coating activated carbon particles with the non infectious bacterium Pseudomonas fluorescens so as not to bnd infectious target bacteria. A limitation to this new activated carbon methodology is our observation that this technique works with certain target organisms but not with others.<P> The objectives of these studies are therefore to (1) to identify bacterial species that are maximally suitable for blocking the surface of activated charcoal for use with a maximum number of food borne pathogenic bacterial species, (2) to identify alternate coating agents such as bentonite in place of Pseudomonas fluorescens, and (3 to develop a universal protocol for all food borne bacterial pathogenic bacteria for direct detection of low numbers (1 to 10 per gram of food) by PCR without enrichment utilizing coated charcoal.
Non-Technical Summary: Some meat products contain potent PCR inibitors that are not completely removed by commercial DNA extraction and purification kits. The purpose of these studies is to develop methodology to facilitate the rapid 3 hour detection and quantification of low numbers of pathogenic bacteria (1 to 10 per gram) in complex food matrices by PCR using specialy treated activated charcoal to remove the PCR inhbitors from food samples. <P> Approach: Particles of activated charcoal have binding capacity on their surface for both charged organic molecules and bacterial cells. Hence bacterial cells will be bound tenaciously to the surface of charcoal particles. In addition, the majority of the binding capacity of activated charcoal resides in the submicroscopic pores. By coating (blocking) the binding sites on the surface of charcoal particles with intact bacterial cells, it is hoped that further binding of target bacterial cells will not occur. The submicroscopic pores will still be available for binding soluble organic molecules from foods that contain potent PCR inhibitors. We will use several approaches. (1) variation in pH from 5.0 to 8.0 when target pathogenic organisms (E. coli O157:H7, L. monocytogenes, Vibrio vulnificus, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli, and various serotypes of Salmonella) are applied to charcoal coated with P. fluorescens, (2) coating the activated charcoal with additional non pathogenic organisms(Acetobacter aceti, Bacillus subtilis, Moraxella immobilis (Moraxealla cryophilus), Pediococcus cerevisiae, Micrococcus luteus, Lactococcus lactis, Sarcina lutea, and a commensulate strain of E.coli and (3) assessing the ability of activated charcoal coated with bentonite to not bind target pathogenic bacteria. The final stage of these studies will be to apply the optimized carbon blocking system to the enumeration by conventional and SYBR green mediated real-time PCR of the various food borne pathogens seeded into various foods such as hamburger, fish tissue, shellfish tissue, and soft cheeses. Conventional and real-time primers and protocols will be from the published literature.