A group-specific PCR assay for the detection of Helicobacteraceae in human gut

Abstract

Background: Enterohepatic Helicobacter species are emerging pathogens, which are increasingly isolated from humans with enteric diseases. Nevertheless, current methods to detect Helicobacteraceae in the human gut have significant limitations.

Methods: Based on 16S-rRNA gene alignments and computer aided primer analysis a set of group-specific PCR primers was designed. The evaluation of the PCR assay was performed using 36 ATCC reference strains and intestinal biopsies from 10 patients with defined gastric Helicobacter pylori status. The amplification products derived from clinical samples were cloned and subsequently analyzed by DNA sequencing. Sensitivity of the PCR-assay was determined by spiking previously negative tested samples with decreasing amounts of Helicobacter DNA.

Results: The analysis of the ATCC reference strains revealed amplification products in all 14 Helicobacter strains and Wolinella succinogenes, 21 other microorganisms representing negative controls did not produce PCR fragments. Four out of the 10 patient-derived samples were positive. Three of them represented H. pylori-derived DNA confirming the gastric H. pylori infection in these patients. In the fourth patient, who was suffering from Crohn's disease, H. pullorum was identified. The sensitivity of the PCR assay was 0.1 pg of Helicobacter-derived DNA representing about 40 bacteria.

Conclusion: The novel PCR assay described here is an important new tool in rapid and sensitive assessment for the presence of Helicobacteraceae in human gut.