Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Optimal methods for detection of Helicobacter pylori from mouse gastric tissue

Real-time quantitative PCR is a sensitive method to assess H. pylori load in infected mice, but there is a need to standardise the preparation of DNA from gastric specimens. In the study, mouse stomachs were homogenised by:

- complete disruption using a blender (Ultra Turrax) or,
- by vortexing with glass beads (1mm diameter).

Each procedure was followed by DNA purification by one of three different protocols - two commercially available kits - Qiagen DNA Tissue kit and Qiagen Stool Kit or a phenol-chloroform extraction method. Homogenisation with glass beads followed by the Tissue kit was found to be most suitable protocol combining high extraction and detection efficiency of 16S rDNA. Analyses of PCR inhibition showed a strong correlation with DNA concentrations and indicated that inhibition was probably due to substances co-purified with DNA rather than excess of mouse non-target DNA.

To overcome PCR inhibition in samples produced by vortexing and the Qiagen Tissue kit, a 10-fold dilution should be applied before the 16S PCR assay. The procedure was validated by quantifying H. pylori DNA in infected mice and allowed accurate detection of H. pylori DNA even in the case of successfully immunised mice. When infection rates are to be determined, it is important to correct the 16S H. pylori DNA quantities with the host DNA quantities determined as total DNA or as mouse GAPDH DNA. Use of this method should allow reproducible, sensitive and accurate detection of H pylori in mouse stomachs.

Reported by

BARTS & THE LONDON NHS TRUST
West Smithfield
EC1A 7BE London
United Kingdom
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