You are here:
QUANTITATIVE MEASUREMENT OF HELICOBACTER PYLORI BY THE TAQMAN FLUOROGENIC PROBE SYSTEM
Citation:
McDaniels, A E., G N. Stelma Jr., AND R A. Haugland. QUANTITATIVE MEASUREMENT OF HELICOBACTER PYLORI BY THE TAQMAN FLUOROGENIC PROBE SYSTEM. Presented at American Society of Microbiology, Los Angeles, CA, May 21-24, 2000.
Impact/Purpose:
1. To identify those pesticides, pathways, and activities that represent the highest potential exposures to children;
2. To determine the factors that influence pesticide exposures to children;
3. To develop methods for measuring multimedia exposures to children, including methods that account for important activities that take place in home, school, and day care settings;
4. To generate data on multimedia pesticide concentrations, pesticide biomarkers, and exposure factors that can be used as inputs to aggregate exposure models for children.
Description:
Culturing of H. pylori from environmental sources continues to be an obstacle in detecting and enumerating this organism. Successful methods of isolation and growth from water samples have not yet been developed. In this study a method involving real tme PCR product detection with a fluorogenic hybridization probe (TaqMan) was evaluated for use in quantifying H. pylori cells. Selection of primer and probe sequences for the ureA gene was performed based on comparative sequence analyses of 16 strains of H. pylori and other Helicobacter species. Samples of serially diluted H. pylori cells spanning a 6-log concentration range were subjected to DNA extraction and TaqMan analysis. Estimated cell quantities in the extracted samples ranged from 20 to 2 x 10-6 based on direct microscopic counts following staining with DAPI. Results from 5 replicate experiments showed a good correlation between TaqMan assay results (expressed as cycle threshold values) and the logarithms of expected cell numbers based on direct counts over the entire cell quantity range tested. Similar results were seen for the two Helicobacter pylori strains. It was concluded that the TaqMan quantitative PCR method has the potential to provide accurate quantification of H. pylori cells in environmental samples.