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DETECTION OF PATHOGENS IN DRINKING WATER (SEER 2)
Impact/Purpose:
The detection of pathogens in drinking water depends largely on traditional approaches, but cellular chemical constituents, i.e., lipids and sugars in cell walls and membranes, are useful as specific markers for microbial species or strains. We propose that it is possible to use chromatographic procedures, including anion-exchange liquid chromatography (AELC), HPLC/MS, GC/MS, and ES/MS/MS, to detect molecules of clinical relevance in water at ng/L or pg/L concentrations. Our primary objectives will be to: (1) develop detection methods based on GC/MS, ES/MS/MS, and AELC for sugar and lipid molecules specific as identifiers for a variety of important pathogenic bacteria that threaten drinking water in the United States; (2) confirm the specificity and sensitivity of each detection method developed; and (3) demonstrate the efficacy of the detection methods with actual drinking water samples under controlled (laboratory) conditions, and in the field.
Description:
Project investigators developed a polymerase chain reaction (PCR)-based technique to detect E. coli 0157:H7 cells in environmental samples using previously reported PCR primers for the specific detection of genes involved in biosynthesis of 0157 polysaccharide and H7 flagella antigens. They also developed a multiplex PCR technique to simultaneously detect animal cells (sheep, cow, human, or horse) and E. coli 0157:H7. PCR primers to detect animal cells were based upon the known DNA sequences of J region and C region of the regulatory D-loop of mitochondrial DNA. E. coli 0157:H7 (ATCC 43894) was used as the pathogen standard for PCR procedure development. E. coli DH5 and non-O157:H7 E. coli strains isolated from cattle feces were used as negative controls. Experimental samples were taken from water troughs of the various animals being studied as possible contamination sources. The E. coli 0157:H7 titer detected by the PCR assay had as few as six cells. Two hundred E. coli 0157:H7 cells were easily detected after 38 amplification cycles. The D-loop primer sets were able to detect and differentiate the presence of human from horse or cattle, but not sheep DNA samples.