A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis

Caroline Blaecher, Eva Bauwens, Alfred Tay, Fanny Peters, Sylvia Dobbs, John Dobbs, André Charlett, Richard Ducatelle, Freddy Haesebrouck*, Annemieke Smet

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

Background: Helicobacter suis is a very fastidious microorganism associated with gastritis, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma in humans. In vitro isolation of this agent from human patients has so far been unsuccessful. Materials and methods: A probe-based real-time PCR (RT-PCR) for the rapid detection of H. suis in gastric biopsies was developed. Secondly, a mouse-passage-based protocol was optimized for isolation of low numbers of viable H. suis bacteria. Mice were inoculated with different numbers of viable H. suis (102-108) and kept for 4 weeks to allow multiplication of this pathogen. Results: The probe-based real-time PCR (RT-PCR) exhibited a high degree of diagnostic specificity and analytical sensitivity, high linear correlations (r2 between 0.995 and 0.999), and high amplification efficiencies (>90%) for H. suis. No cross-reactivity was detected with human, porcine, non-human primate, and murine DNA nor with DNA from other bacteria including Helicobacter spp. and Campylobacter spp. H. suis was successfully re-isolated from the stomach of mice inoculated with at least 104 viable H. suis, using a biphasic medium (pH 5), consisting of Brucella agar with Brucella broth on top, both supplemented with vitox supplement, Campylobacter-selective supplement, amphotericin (5 μg/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5 μg/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli. Conclusion: The methods described above can be implemented for detection or isolation of H. suis from human gastric biopsies.

Original languageEnglish
Article numbere12369
JournalHelicobacter
Volume22
Issue number3
DOIs
Publication statusPublished - Jun 2017

Bibliographical note

Publisher Copyright:
© 2016 John Wiley & Sons Ltd

Keywords

  • Helicobacter suis
  • diagnosis
  • isolation
  • probe-based RT-PCR

Fingerprint

Dive into the research topics of 'A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis'. Together they form a unique fingerprint.

Cite this