Mitochondrial COX2 primer for bovine identification using qPCR: Design and validation

Abstract Beef is a prime livestock product that is prone to adulteration with other relatively lower price species. The occurrence causes losses to consumers and also violates consumer protection law. Quantitative PCR (qPCR) is a molecular approach technology that is suitable for detecting the presence of a broad spectrum of DNA in raw and processed animal-based foods. Mitochondrial DNA (mtDNA) is well known having unique nucleotide sequence which is able to be used as genetic markers in living things. The cytochrome C oxidase subunit II ( COX2 ), a part of mtDNA, can be explored to be biomarker candidate for detection of contamination in various animal products. Therefore, this study was conducted to design and validate specific primer targeting mtDNA COX2 for bovine identification using qPCR. This study designed forward primer (5’-CATGAGCTGTGCCCTCTCTAG-3’) and reverse primer (5’-TAATATAAGCCTGGACGGGA-3’) using the National Center for Biotechnology Information (NCBI) website with accession number GU947020. Moreover, those primers were tested in vitro to optimize reaction condition, and component, and also to evaluate repeatability and sensitivity of the qPCR. Three species DNA, bovine, pig, and chicken were utilized in this study. This study found that the designed primer pair successfully amplified the DNA target in 32 cycles of qPCR, Cq value at 22.48±0.38, Tm at 82.55°C, and 20 ng/μL of bovine DNA. In addition, the limit of detection (LOD) was 2.08 ng/μL and the primer pair specifically attached to bovine DNA. This study concluded that the primer pair targeting mtDNA COX2 can be a strong biomarker candidate for the identification of bovine DNA in various food products.