Application of PCR in Food Biochemistry

For detection of genetically modified organisms, several different methodologies could be employed. Different types of quantitative PCR (qPCR) are used for tracing changes of DNA and/or RNA differing in type of compound and mechanism used for detection (intercalating dyes, primers and probes). Different types of PCR were developed based on specific part of DNA being investigated. Besides PCR methods, several novel methods are employed. Next generation sequencing is used for obtaining information about localization and sequence of insert and its flaking regions. Microarray technology allows for multiple DNAs to be analyzed simultaneously on the chip. Decision-support system that detect and quantify GMO based on the Ct and Tm values and the LOD and LOQ is used in so-called matrix based methods. In so-called “food forensics” parameters like protected designation of origin (PDO) and protected geographic indication (PGI) that serve as an indication of food quality are being determined. In PGI determination several methods can be employed depending from type of molecule or parameters being analyzed, e.g. mass spectrometry for determining isotope ratio, spectroscopy or chromatography How to cite this book chapter: Pavlićević, M. and Vucelić-Radović, B. 2019. Application of PCR in Food Biochemistry. In: Vucelić Radović, B. , Lazić, D. and Nikšić, M. (eds.) Application of Molecular Methods and Raman Microscopy/ Spectroscopy in Agricultural Sciences and Food Technology, Pp. 97–117. London: Ubiquity Press. DOI: https://doi.org/10.5334/ bbj.g. License: CC-BY 4.0 98 Application of Molecular Methods and Raman Microscopy for monitoring changes in lipid profiles etc. For determination of PDO, PCR methods that trace unique sequences, like single nucleotide polymorphisms (SNPs), restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLP), simple sequence length polymorphisms (SSLPs) are used. Novel method called DNA barcoding uses markers (short sequence complementary to target region) that can identify variation among cultivars. Today, in food forensics genomics, proteomics and metabolomics for determining authenticity are used. Both for DNA and RNA extraction, due to “matrix effect” choice of extraction method is of crucial importance for obtaining high amount of non-degraded pure nucleic acid. Thus, although both extraction of DNA and RNA consist of same basic steps (homogenization, lysis and extraction, purification (precipitation or binding), elution or resolubilization), lysis and extraction step will have the most influence on quality of isolated nucleic acid. During DNA extraction it is possible to use either kits (that are developed for specific purpose) or “traditional methods” were organic solvents are used for extraction. Today, for extraction of RNA mostly kits developed for RNA extraction from specific organisms and/or tissue are used. Since RNA is chemically more unstable than DNA it can easily be degraded during sampling and homogenization. For monitoring gene expression, Real Time PCR is used. Design of primer is possible in on-line free software such as Primer3 and Primer3Plus from NCBI or in software that are supplied with Real time PCR apparatus. Analysis of primers (possibility of formation of secondary structure, like hairpins and/or primer-dimmers) can be done in software like Vector NTI and MP primer.