Microfluidic analysis of multiplex
PCR products for the genotyping of
Helicobacter pylori
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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.
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The human pathogen Helicobacter
pylori (H. pylori) is associated
with the development of a variety
of gastro-duodenal diseases, such
as chronic active gastritis, peptic
ulcer disease, gastric cancer, and
gastric mucosa associated lymphoid
tissue (MALT) lymphoma1. It is estimated that about half of
the world's population is infected
with H. pylori. But despite the
high prevalence of infections only
a minority of infected individuals
will develop gastric carcinoma or
MALT lymphoma. Therefore, it is
essential to identify factors that
might determine the possible
sequelae of the H. pylori infection,
such as host and environmental
factors and bacterial virulence
genes.
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The specific H. pylori virulence
genes found to be associated with
possible disease outcome are the
various alleles of the vacA gene,
the presence of the cagA gene as a
marker for a pathogenicity island,
and the allelic variants of the iceA
gene. The vacA gene is present in
all H. pylori strains and encodes
for the vacuolating cytotoxin,
VacA which causes vacuolar
degeneration of gastric epithelial
cells. The vacA gene, although
present in all strains, differs in its
allelic variants in the signal region
and the mid-region of the gene.
The sequence variations in the signal
region are distinguished into vacA s1 and vacA s2 subtypes, and
the mid-region can be distinguished
into m1 or m2 subtypes2.
The allelic variants of the vacA
gene that were detected by nested
PCR were vacAs1/m1, vacAs1/m2
and vacAs2/m2. These vacA gene
variants are associated with different
cytotoxin activities. The cagA
gene is a marker for the presence
of the cag pathogenicity island,
which is a 40 kb locus containing
31 genes associated with the
induction of interleukin 8 secretion
by gastric epithelial cells3.
The allelic variants of the iceA
gene are distinguished into iceA1
or iceA2 and only one of which
being found in all H. pylori strains.
The iceA gene seems to be
induced by contact with epithelium
and associated with increased
mucosal interleukin 8 concentrations4.
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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.
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Material and methods
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Tissues
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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 µm-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 acetate and 50 %
isopropanol.
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PCR
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For H. pylori genotyping, a
sequence specific, nested PCR
approach was performed as
described earlier by Koehler et al.
[5]. Briefly, genomic sub-fragments
of the virulence factors
vacA, cagA, and iceA were amplified
by the combined use of allelespecific
primer sets (vacA s1/s2,
vacA m1/m2, cagA, and iceA 1/2)
in two PCR amplification steps,
each starting with a preincubation
at 95°C for 5 minutes, then followed
by 35 cycles of 1 minute
denaturation at 95 °C, 1 minute annealing at 58 °C and 1 minute
extension at 72 °C. PCR yielded
products with sizes varying from
102 bp to 301 bp.
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Analysis of PCR-products
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PCR products were analyzed using
standard agarose gel electrophoresis
and Lab-on-a-chip technology
on the Agilent 2100 bioanalyzer
using the Agilent DNA 1000
LabChip® kit according to the
manufacturer's instructions.
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Results and discussion
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In a previous study 5 we have
established a nested multiplex
PCR (mPCR) assay in order to
identify the different virulence factors
of Helicobacter pylori, vacA,
cagA and iceA genotypes in routinely
processed gastric biopsies
which are formalin fixed and
paraffin embedded. The nested
mPCR assays with a limited number
of primer sets produced reliable,
distinct amplification products
of all specimens. The combined
PCR assays for the simultaneous
detection of the vacA midregions
m1 and m2, as well as the
combined detection of iceA1 and
iceA2 were routinely performed.
The semi-nested PCR for the
detection of the vacA signal
regions s1 and s2 was usually
combined with the nested PCR for
the cagA gene. The established
mPCR assay permits the simultaneous
analysis of all seven different
diagnostically relevant alleles.
However, tested on a broad spectrum
of diagnostic samples and
analysed on a standard agarose
gel, the assay resulted in PCR fragment
patterns that were difficult
to interpret in some cases.
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To increase analytical precision of
these studies the Agilent 2100 bioanalyzer,
using Lab-on-a-chip technology
was tested to replace slab
gels as the tool for the electrophoretic
separation and analysis
of the mPCR products. First,
we compared the analysis of the
routinely performed combined
PCRs of vacAs1/s2/cagA, of vac-
Am1/m2 (data not shown) and of
iceA1/2 (figure 1) on the Agilent
2100 bioanalyzer with slab gel
results. The microfluidic analysis
of the PCR amplicons of the H.
pylori virulence genes showed and
confirmed the results of our samples
as imaged by slab gel electrophoresis.
Especially the
improved fragment resolution by
the Agilent 2100 bioanalyzer (figure
1B) compared with the slab
gel analysis (figure 1A) and the
representation as an electropherogram
(figure 1C) were helpful in
differentiating DNA fragments,
which differ in size by only 29 bp
as with iceA1 and iceA2 (figure 1).
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Next, the combined analysis of the
five different alleles (vacA s1 or
s2, cagA, vacA m1 or m2) was
investigated (figure 2). Whereas
the detection of the 5 alleles of H.
pylori genotypes was sometimes
difficult when imaging was performed
with slab gel electrophoresis
the imaging of microfluidic
data showed improved analysis of
distinct amplification products.
The gel-like image shows the separation
of vacAs2/vacAm2 in lane 1
and 4 and vacAs1/cagA/vacAm1 in
lane 2 and 3 (figure 2A). The
analysis resulted in calculated
sizes of 98 and 101 bp for the
vacAm2 amplicon and in calculated
sizes of 151 and 148 bp for the
vacAs2 amplicon (expected sizes:
vacAm2: 102 bp and vacAs2: 150
bp, respectively). In addition to the gel like image, the result of
lane 1 and 2 of figure 2A are
displayed as electropherograms
(figures 2B and 2C).
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Finally, combined microfluidic
analysis of all 7 alleles of H.
pylori amplified in 4 different
mPCRs was possible on the chip
after mixing all mPCR reactions
prior to analysis (combination of:
iceA1/A2, vacA s1/s2, cagA, vacA
m1/m2). This is difficult with slab
gel electrophoresis where differences
in the amount of PCR products
cause difficulties in data
interpretation. All seven different
mPCR products within a range of
102-301 bp could be separated
and quantified (figure 3). These
results reveal that Lab-on-a-chip
technology enables the separation
of seven different targets
restricted to a size range up to
300 bp in a combined mPCR
reaction.
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Conclusion
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The results of this study clearly
demonstrate the ability of the Agilent
2100 bioanalyzer to differentiate
the allelic H. pylori variations,
vacAs1/s2/cagA, of
vacAm1/m2 and of iceA1/2, amplified
by mPCR. The high resolution
achieved by the microfluidic
based Lab-on-the-Chip technology
allows to analyze a broad and
expanding panel of virulence and
risk factors. This is of great
importance since an extended
spectrum of prognostic or therapeutically
relevant information
used in H. pylori diagnostics will
now be accessible for routinely
processed simultaneous diagnostics
by means of mRCR assays.
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