Characterization of a polymerase chain reaction-based approach for the simultaneous detection of multiple...

In this study, a polymerase chain reaction (PCR) primer set capable of amplifying a mitochondrial DNA segment of multiple species (cattle, sheep, goats, deer, and elk) whose rendered remains are prohibited from being fed to ruminants was characterized. However, the primer set also amplifies DNA derived from the rendered remains of pigs and horses, which are exempt from the feed ban. PCR amplicons derived from pig DNA have a restriction endonuclease site recognized by Hinf1, while the horse DNA-derived amplicon has a unique restriction endonuclease site recognized by HypCH4III. This "universal" PCR primer produced an amplicon with DNA extracted from dairy feed containing either bovine meat and bone meal or pig blood meal. Enzymatic digestion of the PCR amplicons from these feed samples with Hinf1 resulted in cleavage products only from samples containing pig blood meal. However, Hinf1 digestion of these amplicons was not complete. Further analysis of the pig blood meal with primers specific for bovine or porcine DNA demonstrated the presence of both bovine- and porcine-derived DNA. Enzymatic digestion confirmed these findings. Additional testing was conducted with dry dog food samples labeled as containing either lamb, chicken, turkey, or chicken and fish. The universal PCR primer produced an amplicon only for the dog food containing lamb meal. This paper is the first to describe a simplified approach for the detection of the prohibited species of concern in the feed ban.