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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

Monstein, H J; Olsson, Crister LU ; Nilsson, I; Grahn, N; Benoni, Cecilia LU and Ahrné, Siv LU (2005) In Journal of Microbiological Methods 63(3). p.239-247
Abstract
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... (More)
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. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
Bacterial 16S rDNA, Bacterial profiling, Colorectal biopsy, Helicobacter spp., Multiple displacement amplification, Pyrosequencing, TTGE
in
Journal of Microbiological Methods
volume
63
issue
3
pages
239 - 247
publisher
Elsevier
external identifiers
  • pmid:15935494
  • wos:000233489700003
  • scopus:27744480558
ISSN
1872-8359
DOI
10.1016/j.mimet.2005.03.012
language
English
LU publication?
yes
id
da28ae95-8167-460e-acb0-db4e0458426e (old id 152821)
date added to LUP
2007-07-19 08:53:24
date last changed
2017-07-30 03:46:20
@article{da28ae95-8167-460e-acb0-db4e0458426e,
  abstract     = {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.},
  author       = {Monstein, H J and Olsson, Crister and Nilsson, I and Grahn, N and Benoni, Cecilia and Ahrné, Siv},
  issn         = {1872-8359},
  keyword      = {Bacterial 16S rDNA,Bacterial profiling,Colorectal biopsy,Helicobacter spp.,Multiple displacement amplification,Pyrosequencing,TTGE},
  language     = {eng},
  number       = {3},
  pages        = {239--247},
  publisher    = {Elsevier},
  series       = {Journal of Microbiological Methods},
  title        = {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},
  url          = {http://dx.doi.org/10.1016/j.mimet.2005.03.012},
  volume       = {63},
  year         = {2005},
}