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Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis

In English

Författare

  • H J Monstein
  • Crister Olsson
  • I Nilsson
  • N Grahn
  • Cecilia Benoni
  • Siv Ahrné

Summary, in English

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens. (c) 2005 Elsevier B.V. All rights reserved.

Avdelning/ar

  • Department of Food Technology, Engineering and Nutrition
  • Chronic Inflammatory and Degenerative Diseases Research Unit
  • Gastroenterologi

Publiceringsår

2005

Språk

Engelska

Sidor

239-247

Publikation/Tidskrift/Serie

Journal of Microbiological Methods

Volym

63

Issue

3

Dokumenttyp

Artikel i tidskrift

Förlag

Elsevier

Ämne

  • Clinical Medicine
  • Surgery

Nyckelord

  • Bacterial 16S rDNA
  • Bacterial profiling
  • Colorectal biopsy
  • Helicobacter spp.
  • Multiple displacement amplification
  • Pyrosequencing
  • TTGE

Status

Published

Forskningsgrupp

  • Chronic Inflammatory and Degenerative Diseases Research Unit
  • Gastroenterology

ISBN/ISSN/Övrigt

  • ISSN: 1872-8359