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Rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs by real-time polymerase chain reaction with TaqMan minor groove binder probes.
[gm1 gangliosidosis]
Real-time
polymerase
chain
reaction
(
PCR
)
with
TaqMan
minor
groove
binder
(
MGB
)
probes
was
examined
to
establish
a
rapid
and
reliable
genotyping
technique
for
GM
1
gangliosidosis
in
Shiba
dogs
.
This
technique
was
applied
to
DNA
samples
extracted
from
the
blood
,
umbilical
cord
,
or
postmortem
liver
tissue
specimens
,
and
to
DNA-containing
solutions
prepared
from
blood
and
saliva
that
had
been
applied
to
Flinders
Technology
Associates
filter
papers
(
FTA
cards
)
.
The
amplification
of
the
targeted
sequence
in
all
the
samples
was
sufficient
to
determine
the
genotypes
of
GM
1
gangliosidosis
.
Forty
-
seven
DNA
samples
that
had
previously
been
obtained
from
blood
or
tissue
specimens
of
Shiba
dogs
were
examined
using
this
real-time
PCR
technique
,
and
the
findings
were
consistent
with
the
data
obtained
by
the
earlier
PCR-restriction
fragment
length
polymorphism
(
RFLP
)
assay
.
In
addition
,
the
use
of
this
new
technique
in
combination
with
FTA
cards
for
sampling
could
markedly
shorten
the
time
required
for
genotyping
,
as
well
as
simplify
the
procedure
.
Furthermore
,
in
the
present
study
,
the
results
of
a
previous
epidemiological
screening
of
96
Shiba
dogs
in
the
Czech
Republic
were
rechecked
by
this
real-time
PCR
technique
using
stored
crude
buccal
cell
DNA-containing
solutions
directly
as
DNA
templates
.
The
results
provided
clear-cut
genotyping
in
all
the
samples
although
the
earlier
PCR-RFLP
assay
could
not
determine
the
genotype
in
all
cases
.
In
conclusion
,
this
new
real-time
PCR
technique
is
a
simple
,
rapid
,
and
reliable
choice
for
large
-scale
screening
to
detect
an
abnormal
allele
indicating
GM
1
gangliosidosis
in
Shiba
dogs
.
Diseases
Validation
Diseases presenting
"large-scale screening"
symptom
gm1 gangliosidosis
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