Duplex-real time polymerase chain reaction assay for simultaneous analysis of pork and chicken in sausage products

Abstract

The adulteration of meat-based food is common due to the price difference among raw meat materials. One of the favorite foods commonly consumed by Indonesian and Malaysian societies is chicken sausage, which can be substituted by pork to get economical profits. The objective of this study was to develop a duplex real-time PCR assay using the EvaGreen fluorescence dye for the identification of chicken and pork in sausage products. The method involved the application of chicken (Gallus gallus) and pork (Sus scrofa) specific primers which amplify the small fragments (pork 176 bp and chicken 183 bp) of the mitochondrial D-loop 22 and mt-12s rRNA genes, respectively. DNA was isolated from raw meat materials and reference sausage made from the mixtures of chicken and pork to optimize the assay. The primers used for pork were forward 5’-TCG TAT GCA AAC CAA AAC GCC-3’ and reverse: 5’-ATG CAT GGG GAC TAG CAG TTA-3’, while primers used for chicken were forward: 5’ TGA GAA CTA CGA GCA CAA AC 3’ and reverse: 5’ ACA TTG TGG GAT CTT CTA GGT 3’. Gene products of chicken and pork produced two distinct melting peaks simultaneously at 76.5 and 84.5oC, respectively. The detection limit of duplex-real time PCR analysis of the reference sausage samples was 0.5% for pork and chicken meat in sausage products. The coefficient of variation (CV) of threshold cycles (Ct) for amplification was 6.25%, lower than that required by the Codex Alimentarius Commission. Duplex-real time PCR analysis followed by melting curve analysis offered rapid, sensitive, and specific detection of pork and chicken in sausage products.