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Journal of Agrobiology, 25: 145-148, 2008
Detection of DNA fragments from Roundup Ready
soya in blood of broilers
Řehout V., Hanusová L., Čítek J., Kadlec J.., Hosnedlová B.
Jihočeská univerzita v Českých Budějovicích, Zemědělská fakulta, katedra genetiky, šlechtění a
výživy zvířat
Abstract
Possible transfer of DNA fragments from genetically modified Roundup Ready soybean,
containing transgene for tolerance against herbicides with glyphosate, from fodder into
blood of broilers ROSS 308 was studied. Three feeding experiments were performed.
Totally, 118 blood samples were analyzed.
Detection of DNA fragments was divided into two parts. First, we were looking for DNA
fragments of control gene – lectin. The other part was focus on detection of transgene
fragments in blood samples. Positive results (it means presence of DNA fragment) were
reported in 18 samples. Seven samples were positive for control gene. Transgene
fragments were identified in 11 samples. These results demonstrated possible transfer of
DNA fragments from fodder into blood of broilers.
Introduction
Recently, the need for more and better feed
and food is getting important question for
researchers. It has led to development of new
techniques, which improve properties of plants.
Genetically modified organisms (GMO) are
results one of these techniques - genetic
modification.
Genetically modified organisms (GMO) are
understood to be plants, microorganisms or
animals into which foreign deoxyribonucleic
acid (DNA) coding one or more new genes has
been integrated (Flachowsky et al., 2004). It
means, it goes about target change of genetic
material (structure) in a way that is unavailable
by natural recombination. This provides to
produce organism with required properties, e. g.
tolerance to herbicides, insect or conformation
to unfavourable enviromental conditions.
Extension of GMO in world bears questions
of their safety. Potential risks suggested to be
associated with use of GM are unexpected
gene effects, allergenic potential, antibiotic
resistance, gene flow (Bertoni and Marsan,
2005). The last named, gene flow, belongs to
one of the most discussed topics in this sphere.
It is necessary to recomend, that intake of DNA
or DNA fragments is usual for millions of years.
Flachowsky et al. (2005) observe that the
amount of DNA absorbed with food varies
between 0,1 and 1 gram per day by humans
and includes fragments of plant and animal
genes, degradeted to different degrees, as well
as bacterial DNA. However, DNA and DNA
fragments are degraded by gastric acid and
various enzymes in digestive tract. Of course,
the possibility of absorption intact fragments
cannot be excluded (Bertoni and Marsan,
2005). Jonas et al. (2001) confirm that the
likelihood of transfer and functional integration
of DNA from ingested food by gut microflora
and/or human cells is minimal. The same
findings put Aumaitre (2004), who reviewed
transgene transmission in animal through gut in
low amount. In his opinion, the behavior and
destiny of the exogenous gene (recombinant
DNA) closely parallels that of plant single copy
genes introduced with feed. Their destiny is
degradation in gut (Chowdbury et al., 2003).
Einspanier (2001) describes transfer of
nontransgenic plant genomic DNA into organs
of broilers, whereas sequences of the
transgene were not identified. Some studies
proved presence of recombinant DNA in gut
content of pigs fed by Bt maize (Reuter and
Aulrich, 2003) or in ruminal fluid of sheep fed
maize grains (Duggan et al., 2003).
Among the most important GM crops, grown
in the Czech Republic, belong Roundup Ready
soybean and Bt maize. Roundup Ready
soybean is GMO tolerant to herbicide,
especially herbicides with active substance
glyphosphate. Glyphosphate blocks enzyme 5enolpyruvylshikimate-3-phosphate
synthase
(ESPS) from plants. After treatment with
glyphosphate-herbicide, plant isn´t able to
produce shikimate and perish. Roundup Ready
soybean has incorporated 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene
from Agrobacterium sp., which has low ability to
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Journal of Agrobiology, 25: 145-148, 2008
bind glyphosphate. So plants remain able to
produce shikimate in presence of glyphosphate
and are tolerant to herbicide with glyphosphate
(Petr, 2005).
Material and methods
Roundup Ready soybean line GTS
40 – 3 – 2 was used for studies of transfer of
genes from GMO into blood of broilers. Broilers
ROSS 308 were assigned to 4 treatment
groups, fed by different diet. Diet for the first
group contained Bt maize MON810, for the
second group Roundup Ready soybean and for
the third group both, Roundup Ready soybean
and Bt maize MON 810. The fourth group was
control with GMO free diet. Ten broilers were
selected from each group and organs of these
broilers were collected. Experiment was
repeated threetimes. Totally 120 samples of
blood were collected.
Genomic DNA was isolated from blood
following NucleoSpin Blood (50 prep.) kit
(Macherey-Nagel), according to manufacturer´s
protocols. Two controls of isolation were carried
out. Standard electrophoresis for genome DNA
in 1,5% agarose gel containing ethidium
bromide was used like first control. Second
control was performed by PCR for chicken
growth hormone gene. Primers used to control
PCR were: 5´-ATC CCC AGG CAA ACA TCC
TC-3´(forward) (GCH1F) and 5´-CCT CGA CAT
CCA GCT CAC AT-3´(reverse) (GCH1R). After
presoaking 4 min. at 94 °C, 35 PCR cycles
were carried out each consisting of 30 s at
94 °C, 120 s at 60 °C, and 90 s at 72 °C
(Kuhlein et al., 1997).
Detection of foreign DNA in blood samples
was performed using commercial kit for
detection of GMO GMOIdent Roundup Ready
TM Soy (Eurofins - Gene Scan). This kit
contains premastermix for specific transgene,
RRS for Roundup Ready soybean, and
premastermix for control gene - lectin. Primers
for chicken growth hormone gene were used
like internal control.
Multiplex PCR was used like method for
detections. First, multiplex PCR for control gene
was performed. Reaction mixture was allowing:
19,9 µl premastermix with primers for control
genes, 1 µl primers GCH1R and GCH1F each,
2 µl dNTP´s, 2 µl Taq and 2 µl DNA. PCR
consists of 2 min. presoaking at 94 °C and 50
cycles: 25 s at 94 °C, 30 s at 62 °C and 45 s at
60 °C, following by 3 min. at 72 °C. PCR
products were analysed by electrophoresis in
2% agarose gel, containing ethidium bromide.
Positive and negative control were carried out.
Every sample was analysed threetimes.
Positive control with commercial genomic DNA
and negative control without DNA in mixture
were carried.
After this part of work, multiplex PCR for
transgene detection was performed. There was
used premastermix for specific transgene in
PCR reaction mixture for detection of specific
transgene. All others conditions were the same
like by PCR for control genes.
Results and discussion
Table 1 summarizes amount of performed
analyses. First problem of this work was
connected with isolation DNA from chicken
blood. Although we used commercial kits for
isolation, we had to solve some problems.
Blood of birds has different properties than
blood of mammals and the quality of isolated
genomic DNA was not very high. An other
problem was with higher coagulation of blood.
We changed a manufacturer´s protocol a little
bit. We increased amount of every component
of kit – usually about 10 %. We also had to
isolate every sample twice and some samples
were isolated threetimes. We were able to
isolate DNA from 118 samples from total 120
samples of blood. Quality of DNA isolation was
confirm by electrophoresis and PCR reaction
with primers for chicken growth hormone gene.
Higher number of detections was caused also
by necessity of 3 repeats for every analysis.
The reason of this procedure is to exclude
casualness of results and possibility of human
error.Positive and negative reactions were used
for exclusion of sample contamination. We used
1 positive and 1 negative control for every 5
samples.
Tab. 1 Summary of number of analyses
Blood samples
Samples
Soybean - control
Soybean – transgene
348
325
Number of analyses
Positive and negative
controls
142
136
Total
490
461
Number of
isolations
246
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Journal of Agrobiology, 25: 145-148, 2008
The genomic DNA isolated from the
samples was used as template in PCR
reactions. The first PCR was control reaction
with primers for chicken growth hormone. This
PCR was used for confirmation of ability of
isolated DNA to produce PCR product. PCR
product has length about 1500 bp. All 118
samples of genomic DNA react positive on PCR
with primers GCH1R and GCH1F .
One hundred and eighteen samples of blood
were analysed for detections of presence of
Roundup Read soybean control gene and
transgene. Multiplex PCR was used for these
analyses. This method is based on using two
pairs of primers. One pair is for detection of
appropriate control gene or transgene. An other
pair serve like an internal control and for
elimination of false negative results. In case,
that internal control is not present, reaction can
be regarded as correctly performed and it is
necessary
to
repeat
the
reaction.
Demonstration of multiplex PCR is displayed by
Fig. 1.
First step was detection of control gene for
soybean – lectin. Fragments of lectingene
(110 bp) were found in 7 samples of blood.
Positive detection in all 3 repeats was found by
3 samples. The other 4 samples had positive
reactions just in 1 or 2 repeats. An other part
was focused on detection of transgene of
Roundup Ready soybean. There were reported
11 positive detection of transgene of Roundup
Ready soybean. Length of DNA fragments is
about128 bp. These detection were not
confirmed in all 3 repeats. can not affirm, that
transfer of transgene from fodder into organs of
broilers is possible. Summary of results of both
parts of experiment are summarized in Table 2.
Occurence of DNA fragments in 1 or 2 repeats
of analyses can be explain by lower quality of
isolated DNA.
Tab. 2 Summary of results of detections of control gene and transgene of Roundup Ready soybean in
samples of blood
Number of positive detections
Total
Total
number of
samples
7
11
118
Blood samples
Soybean - control
Soybean - transgene
Positive detections
(in 3 repeats)
3
0
Our results are in concordance with results,
reported by other authors (Doerfler., 2000;
Aeschbacher et al., 2005). Aeschbacher et al.
(2005) reported this transfer by broilers alsoIt
can be confirmed that transfer of DNA
fragments from intestinal tract into organs of
recipient is possible, but not very usual.
Multiplex PCR represent suitable method for
Positive detections
(in 1 or 2 repeats)
4
11
detection of fragments of foreign DNA. This
method is not available to detect, if a
transferred fragment can be functional in the
organism of recipient. Transfer of unfunctional
fragment can be explain by many ways, from
crosscontamination to possibility, that recipients
had some infection (Petr, not published).
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Journal of Agrobiology, 25: 145-148, 2008
This work was financially supported by MSM
6007665806.
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