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.
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.
Monstein
Hans-Jürg
HJ
Molecular Biology Laboratory-LMC/IBK, University Hospital, Lab 01, Floor 09, S-581 85 Linköping, Sweden. hanmo@ibk.liu.se
Olsson
Crister
C
Nilsson
Isabelle
I
Grahn
Niclas
N
Benoni
Cecilia
C
Ahrné
Siv
S
D001483:Base Sequence; D001706:Biopsy; D003092:Colitis / Q000382:microbiology; D003106:Colon / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012007:Rectum / Q000382:microbiology; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S