Evaluation of DNA extraction protocols and real-time PCR-based methods for efficient investigation of pig traces in foods

Abstract

Industrially processed foods are composed of a complex mixture of molecules combined under specific chemical and physical conditions. Besides their native interactions, most of the ingredients included in processed foods are highly transformed through extreme heat variations, grinding, freezing, pH, and pressure fluctuations in order to reach the desired final product. Due to their complex structure and high level of degradation, processed foods are difficult to analyse. Undeclared components are often detected in processed foods, and accurate diagnostic testing is required to protect those with health, cultural, and religious restrictions. Molecular biology techniques involving PCR are most frequently used for determining the authenticity of foods containing derivatives of living organisms. In the present work, we investigated four different DNA extraction protocols of three commercial kits, two different quantitative PCR (qPCR) techniques, and six different primer pairs. We analysed 96 extracts (12 samples from each of the eight products) by SYBR Green-based qPCR using the two most specific and sensitive primer pairs, and compared these results to those obtained with standard commercial kits that use dual dye-labelled probes. Adopting high-efficiency DNA extraction protocols, our findings highlighted the importance of targeting several small regions of the mitochondrial genome to effectively detect small traces of porcine products, and reduce the risk of false-negative results. Adopting these will ensure that consumers can make accurate and informed choices.