A universal primer distinguishable PCR (UP-D-PCR) method for simultaneous identification and differentiation of bovine- and ovine/caprine-derived ingredients in ruminant feeds

摘要

Illegal addition of bovine- and ovine/caprine-derived ingredients into ruminant feeds would increase the risk of spreading transmissible spongiform encephalopathies (TSE). In this study, a nuclear DNA sequence with 18-bp deletion in ovine/caprine genomes was screened for specific detection of bovine- and ovine/caprine-derived ingredients in ruminant feeds. A universal primer distinguishable PCR (UP-D-PCR) method based on these sequence differences was then established, which is capable of simultaneously identifying and distinguishing bovine (125 bp) and ovine/caprine (107 bp) in one reaction, with a sensitivity of 0.1 ng DNA or 1 meat and bone meal (MBM). The specificity of UP-D-PCR was strictly tested with 20 animal species and 3 feed materials, and the stability and reliability were adequately evaluated in different individuals of 4 bovine species and 5 ovine/caprine breeds. The co-detection ability of UP-D-PCR method was further confirmed in binary meat mixture samples. Five commercial feed samples were accurately analyzed by the UP-D-PCR method, showing good applicability. The method developed in this study will provide an accurate, efficient, and low-cost technology to combat illegal addition in ruminant feeds and protect human and livestock health.