Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples.
We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8-ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5' FAM reporter and a 3' TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.
The National Diagnostics Centre, BioResearch Ireland, National University of Ireland, Galway.
S
S
Lahiff
M
M
Glennon
J
J
Lyng
T
T
Smith
N
N
Shilton
M
M
Maher
D000821:Animal Feed; D000818:Animals; D015139:Blotting, Southern; D002417:Cattle / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers; D015342:DNA Probes; D005506:Food Contamination / Q000032:analysis*; D008460:Meat; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA