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A novel isolation protocol and probe-based RT- PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis.
- Source :
- Helicobacter; Jun2017, Vol. 22 Issue 3, pn/a-N.PAG, 11p
- Publication Year :
- 2017
-
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 (10<superscript>2</superscript>-10<superscript>8</superscript>) 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 ( r<superscript>2</superscript> 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 10<superscript>4</superscript> 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 10834389
- Volume :
- 22
- Issue :
- 3
- Database :
- Complementary Index
- Journal :
- Helicobacter
- Publication Type :
- Academic Journal
- Accession number :
- 122918228
- Full Text :
- https://doi.org/10.1111/hel.12369