Development and validation of TaqMan real-time PCR assays for quantification of chicken adulteration in hamburgers

Abstract

Four quantification methods based on absolute (model Ⅰ), relative (model Ⅱ) and normalized approaches (models Ⅲ and Ⅳ) were developed to estimate the percentage of chicken substitution in beef burgers using TaqMan real-time PCR. The cytochrome b (cytb) and 18S ribosomal RNA (18S rRNA) regions were used for the chicken identification and normalization of assay results, respectively. The plotted curves of four models using the reference chicken-beef mixtures represented the linear correlation (0.9962−0.9995), and the acceptable amplification efficiencies (98.60 %–110.12 %) within the range of recommended values. The results of validation plan using the model samples (mixtures and burgers prepared with chicken meat or chicken paste in beef meat) showed the better trueness parameters for model mixtures in models Ⅲ and Ⅳ (−0.12 % to −18.03 %) in comparison with models Ⅰ and Ⅱ (−5.11 % to −43.95 %). Also, an over-estimation of chicken adulteration in model burgers was quantified by models Ⅰ and Ⅱ (with trueness 99.18 %–170.19 %), while the normalized models gave a more accurate estimation of chicken percentage (with trueness −0.26 % to −1.29 %). The developed TaqMan-qPCR assay could reliably quantify 0.01 % of chicken meat in chicken-beef mixtures without any cross-reactivity. Finally, the normalized approaches as validated models recognized the mislabeling in some commercial hamburger brands.