Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products

Quantitative real-time PCR (qPCR) is a modern technique that has been widely used for the detection of species used in meat products. For obtaining accurate and reliable qPCR results, we assessed two common DNA quantification methods for isolated DNA and five quantification approaches for qPCR products. DNA dilution based on spectrofluorometric results showed better qPCR results than those based on spectrophotometry in terms of linear correlation, amplification efficiency, and linear dynamic range. Binary pork-beef mixtures were used to construct standard curves of SYBR Green-based qPCR products using five quantification approaches, and they were validated and compared using in-house pork models. 18S rRNA gene normalization methods showed better trueness (−11.79% to −6.73%) than that of methods using absolute and relative standard curves (−28.52% to −18.64%) in a model burger. These normalized reference methods successfully estimated the quantities of pork meat in the range of 100%-0.01% in commercial beef products.