Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water.
A rapid DNA extraction and quantitative, real time polymerase chain reaction (QRTPCR) analysis method targeting the ureA gene of Helicobacter pylori was evaluated for the measurement of these organisms on membrane filters at levels that might be expected to be found in drinking water samples. No interference was seen from high levels of background organisms and related, non-target species were detected at approximately 4-5 log(10) lower levels of sensitivity than H. pylori 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 a species-specific fluorescent antibody and solid phase cytometry analyses. The mean detection 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 log(10) based on duplicate analyses of the samples. One liter drinking water samples from several locations in the US were inoculated with the same H. pylori cell suspensions used to generate the standard curve and gave measurements that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method. This method may be useful for the rapid screening of drinking water for H. pylori.
McDaniels
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AE
United States Environmental Protection Agency, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268-1314, USA. mcdaniels.audrey@epa.gov
Wymer
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L
Rankin
C
C
Haugland
R
R
D002478:Cells, Cultured; D019085:Fluorescent Antibody Technique, Direct; D016480:Helicobacter pylori / Q000166:cytology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014873:Water Pollutants / Q000032:analysis / Q000302:isolation & purification*
D014873:Water Pollutants