Evaluation of a TaqMan real-time PCR assay for detection of chicken, turkey, duck, and goose material in highly processed industrial feed samples

A TaqMan real-time PCR method based on nucleotide sequence variation in the D-loop and 12S rRNA mitochondrial genes has been developed for the specific detection of chicken, turkey, duck, and goose prohibited material in animal feeds. The assay uses 4 primer/probe sets targeting short species-specific mitochondrial sequences together with a positive amplification control based on the eukaryotic 18S rRNA gene. The applicability of the real-time PCR. assay was assessed through analysis of a batch of industrial feed samples subjected to different rendering temperatures according to European legislation regulations. The chicken-specific real-time PCR system allows a highly sensitive qualitative detection of chicken-derived processed animal protein from different tissue-type origins, even in samples containing 0.1% target and subjected to heat treatments higher than 133 degrees C. On the other hand; turkey, goose, and duck real-time PCR systems also allowed detection of as low as 0.1% target material in binary mixtures (muscle/oat) manufactured using the minimum legal requirements for sterilization temperatures (133 degrees C). Quantification results, based on calibration standard curves, were very reproducible under the experimental conditions tested. However, the quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatment of the feeds, which affect the amount and quality of amplifiable DNA.