Identification of bovine material in porcine spray-dried blood derivatives using the Polymerase Chain Reaction technique

摘要

Due to the widely supported theory of bovine spongiform encephalopathy (BSE) spread in cattle by contaminated animal feeds, screening of feed products has become essential. For many years, manufacturers have used blood and plasma proteins as high quality ingredients of foods for both pets and farm animals. However, in Europe, the Commission Regulation 1234/2003/EC temporally bans the use of processed animal proteins, including blood-derivative products, in feedstuffs for all farm animals which are fattened or bred for the production of food. This regulation has some exceptions, such as the use of non ruminant blood products into the feed of farm fish. Authorization of the re-introduction of these proteins into animal feed formulations, especially non ruminant proteins into the feed for non ruminant farm animals, is expected when adequate control methods to discriminate ruminant proteins exist. Currently, the number of validated methods to differentiate the species of origin for most of the animal by-products is limited. Here we report the development of a rapid and sensitive polyrnerase chain reaction (PCR)-based assay, which allows detection of bovine or porcine specific mitochondrial DNA from spray-dried blood derivate products (plasma, whole blood and red cells), as a marker for bovine contamination in porcine products. Sample extracts, suitable for PCR, were easily and quickly obtained with the commercial PrepMan~(TM) Ultra reagent (Applied Biosystems). To confirm the porcine origin of the samples, primers targeting a specific region of 134 bp of the porcine cvtochrome b coding sequence were designed (cytbporcl-F and cytbporc2-R). Previously published PCR primers (L8129 and H8357), specific for a 271 bp fragment of the bovine mitochondrial ATPase 8-ATPase 6 genes, were chosen to accomplish amplification of bovine DNA. The limit of detection (LOD) of the bovine PCR assay was at least of 0.05% (v/v) of bovine inclusion in spray-dried porcine plasma or red cells fraction. In dried whole bloodsamples, sensitivity of the method was found to be at least of 0.1 % (v/v). Since the method described here exhibits high specificity and sensitivity and it is rapid, simple and consistent, it could be successfully utilized as a routine control assay toevaluate the presence of bovine materials in spray-dried blood products.