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EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER
Citation:
MCDANIELS, A. E., L. J. WYMER, C. C. RANKIN, AND R. A. HAUGLAND. EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 39(19):4808-4816, (2005).
Impact/Purpose:
To determine the sensitivity and precision of an inhouse real time probe and primers for Helicobacter pylori.
To determine whether drinking water from different geographical areas will contain inhibitors that will prevent or reduce the detection of Helicobaacter pylori.
Description:
Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.
Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR) analysis method was evaluated for the measurement of Helicobacter pylori cells on membrane filters at concentrations that might be expected to be found in drinking water samples. A QPCR assay utilizing primers and a TaqManTM hybridization probe targeting the ureA gene of H. pylori was developed for the method. Related, non-target species were detected at approximately a 5 log10 lower level of sensitivity by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with species specific fluorescent anti-bodies and solid phase cytometry analyses. The mean sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of +/- 0.57 log10 based on duplicate analyses of individual samples. One liter drinking water samples from several diverse locations in the U.S. all tested negative by the method. Filtrates of additional one liter portions of these samples spiked with the same H. pylori cell suspensions used for the standard curve samples gave results that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method.
Conclusions: The QPCR analysis method, described in this study, can reliably detect as few as 40 H. pylori cells from membrane filtrates of drinking water samples and can determine the numbers of these cells within approximately a 1 log10 interval based on analyses of individual samples.
Significance and Impact of the Study: This method may be useful for the rapid screening of drinking water in the USA for H. pylori.
