Development, evaluation, and peer verification of a rapid real-time PCR method for the detection of animal material

Four real-time PCR assays that can be used with U.S.- and European Union-rendered materials to detect three ruminant species (bovine, caprine, and ovine) and a select set of avians (chicken, goose, and turkey) were developed. This method was evaluated against stringent acceptance criteria previously developed by the U.S. Food and Drug Administration, Center for Veterinary Medicine's Office of Research. Acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% meat and bone meal (MBM) was required, consistent with the sensitivity of the validated PCR-based method currently used by the U.S. Food and Drug Administration as an aid in enforcement of the Agency's feed ban. PCR primer specificity was determined by using a panel of DNA samples derived from 16 different animal species. The method is able to detect 0.1% rendered material in complete feed in less than 1.5 h of total assay time, a significant improvement over the current method, which requires 7 to 8 h for completion. The real-time assay for the detection of animal material passed stringent acceptance criteria for sensitivity, selectivity, and specificity. The method also passed ruggedness, real-time platform, and second analyst trials. Two external laboratories participating in a peer-verification trial demonstrated 100% specificity in identifying bovine MBM, ovine MBM, or caprine meat meal, while exhibiting a 0.6% rate of false positives. These results demonstrated that this method was capable of being used by other laboratories.