Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs.
This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.
Bellagamba
Federica
F
Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare (VSA), Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Italy. federica.bellagamba@unimi.it
Comincini
Sergio
S
Ferretti
Luca
L
Valfrè
Franco
F
Moretti
Vittorio M
VM
D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002645:Chickens / Q000235:genetics; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D006041:Goats / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011312:Pressure; D017096:Prion Diseases / Q000517:prevention & control / Q000635:transmission; D011328:Prions / Q000032:analysis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012756:Sheep / Q000235:genetics; D013045:Species Specificity; D013552:Swine / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D011328:Prions