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Microfluidic analysis of multiplex,,,PCR products for the genotyping of,,,Helicobacter pylori

and associated with increased mucosal interleukin 8 concentrations4.
In our studies5, we have found that certain H. pylori subtype combinations of the vacA, cagA and iceA genes possess a differentiating and predictive value for the development of gastric adenocarcinoma and MALT lymphoma. We have established a multiplex PCR analysis to detect and distinguish the different H. pylori genes and their allelic variants from DNA extracted from paraffin wax embedded tissues. The use of template DNA derived from formalin fixed and paraffin wax embedded tissue for the mPCR restricts the size of the PCR fragments to a maximum of about 300 bp due to fragmentation and poor DNA quality from such tissue. The primer sets used in the mPCR for the detection of the different H. pylori virulence genes generated amplification products with a maximum length of 301 bp. The combined use of primer sets in the mPCR was possible because each primer set required an annealing temperature of 58 C and all PCR products were distinguishable by size on slab gel electrophoresis. In order to obtain more reproducible and standardized results, a microfluidic based analytical platform, the Agilent 2100 bioanalyzer, using lab-on-a-chip technology6 was tested to replace slab gels as the tool for the electrophoretic separation of the mPCR products.
Material and methods
Tissues
The paraffin embedded and formalin fixed gastric tissues from chronic gastritis, gastric adenocarcinoma and gastric MALT lymphoma specimen were recruited from the archives of the Institutes for Pathology of the University of Mainz (Germany) and Cologne (Germany). Three 5-7 m-sections of each specimen were de-waxed in xylene and the nucleic acids were extracted by phenol/chloroform followed by precipitation in 300 mM sodium acetat
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