Multiple displacement amplification of isolated DNA from human gallstones: molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis.
BACKGROUND
Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp.


METHODS
DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions.


RESULTS
Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones.


CONCLUSIONS
We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens.
Nilsson
Isabelle
I
Division of Biomedicine and Surgery, Faculty of Health Sciences, University Hospital, S-581 85, Linköping, Sweden.
Shabo
Ivan
I
Svanvik
Joar
J
Monstein
Hans-Jürg
HJ
D000328:Adult; D000368:Aged; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal; D005260:Female; D042882:Gallstones / Q000382:microbiology*; D016998:Helicobacter / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016481:Helicobacter Infections / Q000382:microbiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S