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

Abstract

The 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(R) 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.