Microfluidic analysis of multiplex,,,PCR products for the genotyping of,,,Helicobacter pylori
Gastric infection with Helicobacterpylori (H. pylori) is the majorcause of chronic active gastritisand is associated with the pathogenesisof peptic ulcer and gastriccarcinoma. Recent studies using amultiplex PCR (mPCR) basedapproach distinguished differentall
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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