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Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Östergötland.
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Microbiology.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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1998 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 47, no 8, 695-704 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.

Place, publisher, year, edition, pages
1998. Vol. 47, no 8, 695-704 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79462DOI: 10.1099/00222615-47-8-695PubMedID: 9877190OAI: oai:DiVA.org:liu-79462DiVA: diva2:542500
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Identification and characterisation of bacteria based on 16S rDNA techniques with special reference to Helicobacter pylori in the gastro-intestinal tract
Open this publication in new window or tab >>Identification and characterisation of bacteria based on 16S rDNA techniques with special reference to Helicobacter pylori in the gastro-intestinal tract
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The overall aim of this study was to establish molecular techniques for the detection and identification of "difficult to grow" - bacteria in mixed bacterial populations in clinical samples without the need for culturing procedures.

Material and Methods: Thirty-nine strains of Mobiluncus were used as a model system for phylogenetic classification of fastidious bacteria based on 16S ribosomal DNA (rDNA) sequences. To test the application of the 16S rDNA broad range PCR concept for detecting bacteria clinically, urine samples spiked with Chlamydia trachomatis elementary bodies and real urine test samples from 12 C. trachomatis positive and negative male volunteers were tested in a semiblind manner against routine procedures. Furthermore, gastric biopsy samples from 22 individuals (13 defined as having Helicobacter pylori-associated gastritis, and 9 defined as normal controls) were evaluated for the presence of Helicobacter 16S rDNA and the virulence genes cagA, vacA, and ureA. PCR products were also applied to temporal temperature gel electrophoresis (TTGE) gels for profiling the microbial flora. Finally, intestinal biopsies from 22 patients (11 diagnosed as Crohn's disease (CD), and 11 non-CD patients) were investigated using probes targeting potential pathogens that have been suggested to be involved in CD.

Results: We were able to confirm the current species designation of Mobiluncus, although the results did not support the division of M. curtisii into subspecies. For the detection of C. trachomatis in urine, the in-house system was shown to be as sensitive as a commercially available PCR system. The search for Helicobacter pylori in gastric biopsies revealed the presence of Helicobacter DNA in 20 of 22 individuals. The molecular techniques were apparently too sensitive compared with routinely used techniques. TTGE revealed a complex microbial flora both in the normal control group and in the gastritis group, with dominance of Helicobacter in the gastritis group. The present results might lend support to the hypothesis that Helicobacter are indigenous biota of the human stomach. cagA was amplified in all Helicobacter positive specimens. None of the specimens in the control group carried a H. pylori type strain identical vacA genotype. ureA negative mutants were also found in this group. The 16S rDNA sequence data might also indicate phylogenetic heterogeneity. The mixed bacterial flora found in CD inflammatory lesions is consistent with the idea that the enteric microflora enters primary lesions, where secondary invaders may elicit chronic inflannnatory response.

Conclusion: This thesis has demonstrated the usefulness of 16S rDNA based techniques for detection, identification, and characterisation of individual bacterial pathogens as well as for profiling of mixed bacterial flora in clinical specimens.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 112 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 622
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28646 (URN)13802 (Local ID)91-7219-579-7 (ISBN)13802 (Archive number)13802 (OAI)
Public defence
2000-04-28, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-01Bibliographically approved

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Borch, KurtJonasson, JonMårdh, SvenPetersson, FredrikMonstein, Hans-Jürg

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