title: A competitive polymerase chain reaction-based approach for the identification and semiquantification of mitochondrial DNA in differently heat-treated bovine meat and bone meal.
abstract: The risk of bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived proteins in the diet of farm animals. Currently, the EU's officially recommended method for controlling meat and bone meal (MBM) in animal feed is the microscopic method, which involves the identification of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)-based assay can be used for the detection of taxon-specific DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 133 degrees C at 300 kPa for 20 min. Here we investigate the relationship between DNA degradation, PCR amplification, and MBM heat treatment. With a competitive PCR-based approach, we compare the amplification efficiency of bovine mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive DNA is used as an internal control for both DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for amplification was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare amplification efficiency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment.
authors.affiliation: Dipartimento di Biologia, Universita' degli studi di Roma Tor-Vergata, Viale della Ricerca Scientifica, 00133 Rome, Italy. frezza@uniroma2.it
authors.forename: Domenico
authors.initials: D
authors.lastname: Frezza
authors.forename: Marco
authors.initials: M
authors.lastname: Favaro
authors.forename: Gabriele
authors.initials: G
authors.lastname: Vaccari
authors.forename: Christoph
authors.initials: C
authors.lastname: von-Holst
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Giambra
authors.forename: Elke
authors.initials: E
authors.lastname: Anklam
authors.forename: Daniela
authors.initials: D
authors.lastname: Bove
authors.forename: Piero A
authors.initials: PA
authors.lastname: Battaglia
authors.forename: Umberto
authors.initials: U
authors.lastname: Agrimi
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Brambilla
authors.forename: Paolo
authors.initials: P
authors.lastname: Ajmone-Marsan
authors.forename: Marco
authors.initials: M
authors.lastname: Tartaglia
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D002417:Cattle / Q000235:genetics*; D053938:DNA Fragmentation; D004272:DNA, Mitochondrial / Q000032:analysis / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D016643:Encephalopathy, Bovine Spongiform / Q000517:prevention & control / Q000635:transmission; D005506:Food Contamination / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial