Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water.
ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.
Article: Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water.[show abstract] [hide abstract]
ABSTRACT: 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.Water Research 12/2005; 39(19):4808-16. · 4.86 Impact Factor