Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors

Journal of Microbiological Methods
Lurdes MonteiroF Mégraud

Abstract

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA sample...Continue Reading

Citations

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