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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 145 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 246 146 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). 147 Journal of Agrobiology, 25: 145-148, 2008 This work was financially supported by MSM 6007665806. Literature Aeschbacher, K.; Messikommer, R.; Meile, L.; Wenke, C. (2005): Bt176 corn in poultry nutrition: physiological characteristics and fate of recombinant plant DNA in chickens. Poultry Science, 84 (3), 385-394. Aumaitre, A. (2004): Safety assessment and feeding value for pigs, poultry and ruminant animals of pest protected (Bt) palnts and herbicide tolerant (glyphosate, glufosinate) plants: Interpretation of experimental results observed worldwide on GMP. 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