Detection of plant species-specific dna (barley and soybean) in blood, muscle tissue, organs and gastrointestinal contents of rabbit.
Keywords:rabbit, soybean meal, barley grain, DNA fate, real time PCR
AbstractThe aim of this study was to detect plant DNA sequences from low copy number genes of barley grain and soybean meal, the latter being subjected to solvent extraction process, in blood, liver, kidney, spleen, muscle tissue and digesta (duodenum, caecum and faeces from rectal ampulla) of rabbits. For fattening, Hyla rabbits (20 males and 20 females) were fed a diet including barley grain (15%) and soybean meal (12%). Animals were slaughtered at 74 d of age (2 ± 0.2 kg live weight) and samples collected from each animal. The quality of each DNA sample was verified using the UNIV P/Q primers used to amplify a mammalian specifi c portion of mtDNA 16S rRNA gene. The presence of plant DNA was subsequently ascertained on the same DNA samples, as well as on barley and soybean (control). Two classes of plant DNA sequences were monitored via real-time PCR, using SYBR® Green I Dye: a high copy number chloroplast gene (trnl) and a low copy number specific for barley (metal-dependent hydrolase-like protein) and soybean (lectin) genes. Melting curve analysis was used to identify the PCR products. The chloroplast fragment detection frequency was higher (P<0.01) in muscle (90%), liver (80%), kidney (80%) and spleen (80%) than in blood (40%) and digesta samples. In the latter, chloroplast DNA was found in 40 and 30% of duodenum and caecum contents respectively, and in 30% of faeces. The specificity of the amplicons obtained was checked by sequencing and annotation. In the samples positive for chloroplast fragments, the frequency of detection of barley specific sequence was higher (P<0.01) in liver (62.5%), kidney (62.5%), spleen (62.5%) and digesta (100%) than in blood (25%) and muscle (22.2%) samples. The soybean lectin gene was not detected in animal samples, although it was seen in plant samples. Results confirm that, except for gastrointestinal tract (GIT), plant single copy genes are more difficult to identify in animal samples.
Aeschbacher K., Messikommer R., Meile L., Wenk C. 2005. Bt176 corn in poultry nutrition: physiological characteristics and fate of recombinant plant DNA in chickens. Poult. Sci., 84: 385-394.
Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res., 25: 3389-3402.
Association of Official Analytical Chemists, AOAC. 2000. Official Methods of Analysis. 17th ed. AOAC, Washington, DC.
Artim L., Charlton S., Dana G., Faust M., Glenn K., Hartnell G., Hunst P., Jennings J., Shillito R. 2001. Animal performance trials with bt crops. In Proc.: 4th Pacific Rim Conference - Biotechnology of Bacillus thuringiensis and its environmental impact. Australian National Universsity, Camberra, Australia. Nov 1-15, 2001. S: 61 (abstract).
Benedich A. 1987. Why do chloroplasts and mitochondria contain so many copies of their genome? Bioessays, 6: 279-282.
Chen Y., Wang Y., Ge Y., Xu B. 2005. Degradation of endogenous and exogenous genes of Roundup-Ready soybean during food processing. J. Agric. Food Chem., 53: 10239-10243
Chiter A., Forbes J.M., Blair G.E. 2000. DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food. FeBS Lett., 481: 164-168.
Chowdhury E. H., Mikami O., Nakajima Y., Kuribara H., Hino A., Suga K., Hanazumi M., Yomemochi C. 2003. Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLinkTM CBH351. Vet. Hum. Toxicol., 45: 95-96.
Duggan P.S., Chambers P.A., Heritage J., Forbes J.M. 2003. Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Br. J. Nutr., 89: 159-166.
Einspanier R., Lutz B., Rief S., Berezina O., Zverlov V., Schwarz W., Mayer J. 2004. Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize. eur. Food Res. Technol., 218: 269-273.
Flachowsky G., Chesson A.. Aulrich K. 2005. Animal nutrition with feeds from genetically modified plants. Arch. Anim. Nutr., 59: 1-40.
Forbes J.M., Blair G.E., Chiter A., Perks S. 1998. Effect of feed processing conditions on DNA fragmentation. U.K. MAFF Report CS0116.
Klaften M., Whetsell A., Webser J., Grewal R., Fedyk E., Einspanier R., Jennings J., Lirette R., Glenn K. 2004. Animal biotechnology: challenges and prospects. In: ACS Symposium Series (ed. M.M. Bhalgat, W.P. Ridley, A.S. Felsot and J.N. Seiber). American Chemical Society, Washington, DC, vol. 866, pp.: 83-99.
Kuribara H., Shindo Y., Matsuoka T., Takubo K., Futo S., Aoki N., Hirao T., Ariyama H., Goda Y., Toyoda M., Hino A. 2002. Novel reference molecules for quantitation of genetically modified maize and soybean. J. of AOAC Int., 85: 1077-1089.
Mazza R., Soave M., Morlacchini M., Piva G., Marocco A. 2005. Assessing the transfer of genetically modified DNA from feed to animal tissues. Trans. Res., 14: 775-784.
McAllan A.B. 1980. The degradation of nucleic acids in and the removal of breakdown products from the small intestines of steers. Br. J. Nutr., 4: 99-112.
McAllan A.B. 1982. The fate of nucleic acids in ruminants. Proc. Nutr. Soc., 41: 309-317.
Nemeth A., Wurz A., Artim L., Charlton S., Dana G., Glenn K., Hunst P., Jennings J., Shilito R., Song P. 2004. Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA. J. Agric. Food Chem., 52: 6129-6135.
Netherwood T., Martín-Orúe S.M., O’Donnell A.G., Gockling S., Graham J., Mathers J.C., Gilbert H.J. 2004. Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat. Biotechnol., 22: 204-209.
Phipps R.H., Deaville E.R., Maddison B.C. 2003. Detection of transgenic and endogenous plant DANN in rumen fluid, duodenal digesta, milk, blood, and faeces of lactating dairy cows. J. Dairy Sci., 86: 4070-4078.
Sawyer J., Wood C., Shanahan D., Gout S., McDowell D. 2003. Realtime PCR for quantitative meat species testing. Food Cont., 14: 579-583.
Terzi V., Infascelli F., Tudisco R., Russo G., Stanca A.M., Faccioli P. 2004. Quantitative detection of Secale cereale by real-time PCR amplification. Lebensm.-Wiss. u.-Technol., 37: 239-246.
Tudisco R., Infascelli F., Cutrignelli M.I., Bovera F., Morcia C., Faccioli P., Terzi V. 2006a. Fate of feed plant DNA monitored in water buffalo (Bubalus bubalis) and rabbit (Oryctolagus cuniculus). Liv. Sci., 105: 12-18.
Tudisco R., Lombardi P., Bovera F., D’Angelo D., Cutrignelli M.I., Mastellone V., Terzi V., Avallone L., Infascelli F. 2006b. Genetically modified soybean in rabbit feeding: detection of DNA fragments and evaluation of metabolic effects by enzymatic analysis. Anim. Sci., 82: 193-197.
Tudisco R., Cutrignelli MI., Bovera F., Calabrò S., Piccolo G., D’Urso S., Infascelli F. 2007. Influence of pellet process of concentrate on the fate of feed plant DNA in the rabbit. Vet. Res. Comm., 31 (suppl. 1): 409-412.
This journal is licensed under a "Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)".