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

Background: 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. Scope and approach: 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.