Evaluation of DNA extraction methods from mouse stomachs for the quantification of H. pylori by real-time PCR.
Real-time PCR methods have recently been developed for the quantification of Helicobacter pylori from infected mouse stomachs. However, the extent to which results is affected by the efficiency of different methods of DNA extraction and the degree of inhibition of the subsequent PCR have largely been ignored. In this study, mouse stomachs were processed using two homogenisation methods: complete disruption using a blender and homogenisation by vortexing with glass beads. Each procedure was followed by DNA purification by three different protocols-two commercially available kits-Qiagen DNA Mini Tissue kit and Qiagen Stool Kit and a phenol-chloroform extraction method. PCR inhibition was assessed by screening for mouse DNA and for H. pylori DNA after spiking stomach extracts with H. pylori 16S rDNA. PCR inhibition was found to be lower in DNA samples prepared by vortexing and processed by column kits. Validation of procedures was performed by quantification of H. pylori DNA and mouse DNA in infected mouse stomachs. Homogenisation with glass beads followed by the Qiagen Tissue kit was found to be the most suitable protocol combining high extraction and detection efficiency of 16S rDNA in the presence of a mouse DNA background.
Roussel
Yvonne
Y
Department of Microbiology and Virology, Barts and the London NHS Trust St. Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, United Kingdom.
Wilks
Mark
M
Harris
Andrew
A
Mein
Charles
C
Tabaqchali
Soad
S
D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D013047:Specific Pathogen-Free Organisms; D013272:Stomach Diseases / Q000382:microbiology*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S