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

Gastric infection with Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and is associated with the pathogenesis of peptic ulcer and gastric carcinoma. Recent studies using a multiplex PCR (mPCR) based approach distinguished different allelic variants that are associated with different stages of H. pylori virulence. H. pylori genotyping on gastric tissues, routinely processed in diagnostic pathology, revealed that certain combinations of virulence subtypes of different H. pylori sub-strains are associated with gastric carcinoma and MALT lymphoma. The use of formalin fixed samples as the source for template DNA for mPCR restricts the size of the PCR fragments to 300 bp, due to poor DNA quality. For the analysis of multiple bands of a mPCR experiment, the data acquisition from slab gels is often inconvenient in terms of sizing, resolution, and data accuracy. To improve accuracy and reproducibility of the measurements, the Agilent 2100 bioanalyzer using Lab-on-a-chip technology was tested to replace slab gels as the tool for the electrophoretic separation of the mPCR products. First analyses revealed an improved fragment resolution, sizing accuracy and reproducibility. A mPCR reaction covering five different alleles that are involved in H. pylori pathogenesis was analyzed and all fragments were separated and quantified. The analysis of a mixture of all mPCR reactions including all involved alleles showed the separation of seven different PCR fragments in the range of 102 to 301 bp, thereby expanding the spectrum of prognostic or therapeutically relevant information used e.g. in H. pylori diagnostic.
The human pathogen Helicobacter pylori (H. pylori) is associated with the development of a variety o
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