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Abstract
A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.
Acknowledgements
The authors are indebted to Dr R. Margry from CCL-Nutricontrol (the Netherlands) for kindly providing the industrial feed samples analysed in this study. This work was supported by the Programa de Vigilancia Sanitaria (2009/AGR/1489) of the Comunidad de Madrid (Spain) and by a project (AGL2010/15279) from the Ministerio de Ciencia e Innovación (Spain). Nicolette Pegels is recipient of a fellowship from the Ministerio de Educación (Spain).