Basic principles for developing real-time PCR methods used in food analysis: A review

The increased interest in global food fraud has led to the development of a number of advanced methods, among which real-time polymerase chain reaction (PCR) currently plays an integral role in food authentication. However, the lack of standard parameters for the development and validation of real-time PCR methods hampers their utilization across different laboratories and conditions, leading to inconsistent results. This review summarizes and assesses different methods presented throughout a large number of scientific papers, including DNA extraction, primer design, and quantification (or qualification) as well as parameters for the development and validation of real-time PCR methods in food analysis. Key Findings and Conclusions: Inhibitors in DNA extracts can cause decreased PCR sensitivity and false negative results; thus, the use of PCR inhibition and amplification controls (e.g., the 18S ribosomal RNA gene) is essential for obtaining accurate real-time PCR results. In quantitative real-time PCR methods, the results obtained using species-specific systems need to be normalized by using reference systems for the improvement of their accuracy. Therefore, this review will provide researchers with a beneficial guide for the development of real-time PCR methods in a harmonious manner and contribute to an enhanced applicability of the methods developed. •Different methods and criteria used for real-time PCR for food analysis are reviewed.•Use of various PCR inhibition and amplification controls is discussed in detail.•Different quantification and normalization methods are compared and assessed in depth.•Method acceptance parameters are suggested for reliable real-time PCR results.