Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors.
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 samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.
Monteiro
L
L
Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, Bordeaux, France.
Gras
N
N
Vidal
R
R
Cabrita
J
J
Mégraud
F
F
D000328:Adult; D000942:Antigens, Bacterial / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D004032:Diet; D005243:Feces / Q000382:microbiology*; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007124:Immunoenzyme Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013272:Stomach Diseases / Q000175:diagnosis / Q000382:microbiology*
D000942:Antigens, Bacterial; D004269:DNA, Bacterial