PCR detection of bovine mitochondrial DNA derived from meat and bone meal in feed.
Because bovine meat and bone meal (MBM) is thought to be a major source of bovine spongiform encephalopathy, we developed a PCR-based method for detection of bovine MBM in animal feed. We isolated bone particles from feed containing bovine MBM using a separation technique based on specific gravity and then washed bone particles with sodium hypochlorite solution and an EDTA-proteinase K solution. The mitochondrial DNA was extracted from bone particles and amplified using PCR with cattle-specific primers. Bovine DNA was not detected in a milk replacer containing dried skim milk and dried whey, but bovine DNA was detected in the milk replacer that was mixed with bovine MBM. Other cattle-derived materials in feeds did not interfere with the selective detection of bovine MBM. This method allowed detection of bovine mitochondrial DNA in feed with 0.1% added bovine MBM. When the treatment with sodium hypochlorite was excluded, bovine DNA derived from MBM could not be distinguished from bovine DNA derived from other bovine materials. However, the exclusion of this treatment improved the detection limit of bovine MBM in feed. This method appears suitable for the selective detection of bovine MBM in feed.
Toyoda
Atsushi
A
College of Agriculture, Ibaraki University, Ibaraki 300-0393, Japan. atoyoda@mx.ibaraki.ac.jp
Nakajo
Mitsuru
M
Kawachi
Hiroyuki
H
Matsui
Tohru
T
Yano
Hideo
H
D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D002417:Cattle / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000032:analysis*; D016643:Encephalopathy, Bovine Spongiform / Q000517:prevention & control / Q000635:transmission; D005506:Food Contamination / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial