title: Real-time polymerase chain reaction approach for quantitation of ruminant-specific DNA to indicate a correlation between DNA amount and meat and bone meal heat treatments.
abstract: The use of ruminant-derived proteins in ruminant feeds has been banned in both the European Union and the United States to prevent further spread of bovine spongiform encephalopathy. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in feed. We developed a quantitative real-time polymerase chain reaction assay for the quantification of ruminant-specific DNA as index of protein content. The assay is based on the amplification of a 117 base pair mitochondrial 16S rRNA DNA gene fragment and an internal positive control (IPC). The use of an IPC permits compensation for differences in DNA extraction efficiency and avoids the occurrence of false-negative results. We demonstrated a decrease in target DNA amount with a difference of 2 logs between meat and bone meal (MBM) treated at 133 degrees and 145 degrees C. Such a difference indicates that bias could occur when DNA-based methods are used for quantitation purposes. Risk management could benefit from future efforts concerning validation of the method for MBM detection in feedstuff and safety evaluation of the use of animal-derived proteins in animal nutrition.
authors.affiliation: Istituto Superiore di Sanità, Department of Food Safety and Veterinary Public Health, Rome, Italy. barbara.chiappini@iss.it
authors.forename: Barbara
authors.initials: B
authors.lastname: Chiappini
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Brambilla
authors.forename: Umberto
authors.initials: U
authors.lastname: Agrimi
authors.forename: Gabriele
authors.initials: G
authors.lastname: Vaccari
authors.forename: Henk J M
authors.initials: HJ
authors.lastname: Aarts
authors.forename: Gilbert
authors.initials: G
authors.lastname: Berben
authors.forename: Domenico
authors.initials: D
authors.lastname: Frezza
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Giambra
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D001688:Biological Products; D002417:Cattle; D004247:DNA / Q000032:analysis*; D006358:Hot Temperature; D008460:Meat / Q000032:analysis; D008903:Minerals / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D012418:Ruminants / Q000145:classification* / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D001688:Biological Products; D008903:Minerals; D012336:RNA, Ribosomal, 16S; D004247:DNA; C027762:bone meal