Sensitive PCR Analysis of Animal Tissue Samples for Fragments of Endogenous and Transgenic Plant DNA

Abstract

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

Fate of transgenic DNA and evaluation of metabolic effects in goats fed genetically modified soybean and in their offsprings

“…In dairy cows fed GM soybean the chloroplastid sequence was found in leucocytes but not in milk (Klotz and Einspanier, 1998). In other trials carried out with dairy cows fed GM soybean meal and maize (Phipps et al, 2003;Nemeth et al, 2004) or goats fed GM maize (Rizzi et al, 2008), the chloroplast DNA fragments were detected in milk, but no transgenic fragments were found.…”

“…In such a way, a total of six samples for each kid tissue and organ were obtained. In addition, as suggested by Nemeth (2004), negative (buffer only) control to each set of DNA extraction was included to monitor for any contamination that could have occurred during the DNA extraction procedure. The DNA was quantified by spectrophotometer (Thermo Electron Corporation, Waltham, MA, USA) according to standard molecular techniques (Sambrook et al, 1989).…”

“…On this basis, a number of GM products have been approved for human consumption, but concerns over safety persist in the public, mainly regarding either the detection of transgenic plant genes and proteins in animal systems or allergenicity and toxicity of GM plants. Furthermore, some authors have shown the possible survival of recombinant plant DNA fragments after digestion, although health implications as well as biological relevance of data are still far from being confirmed (Nemeth et al, 2004;Mazza et al, 2005;Sharma et al, 2006). The absorption of plant DNA across the intestinal barrier is a natural event, as demonstrated by the detection of endogenous plant genes in several animal tissues and products (Reuter and Aulrich, 2003;Tony et al, 2003;Tudisco et al, 2006b).…”

RETRACTED: <i>Gamma-Glutamyl Transferase Activity in Kids Born from Goats Fed Genetically Modified Soybean</i>

“…All the extracts (in duplicate) were kept at -20˚C until further analysis. In addition, negative (buffer only) control to each set of DNA extraction was included [8]. DNA concentration and purity was determined by using a Biophotometer (Eppendorf, Hamburg, Germany) and PCR amplifications were performed on a Gene Amp PCR System 2400 (Applied Biosystems, Foster City, CA) and carried out as described previously [4].…”

Detection of plant species-specific dna (barley and soybean) in blood, muscle tissue, organs and gastrointestinal contents of rabbit.

“…Indeed, endogenous (native) plant genes have been detected in tissues and products from poultry (Aeschbacher et al, 2005), swine (Mazza et al, 2005) and ruminants (Nemeth et al, 2004).…”