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.
Highlights
Chicken sausages are one of the commonly consumed meat-based products worldwide
The raw meats used in this study namely pork, chicken, beef and others are purchased from local markets around Yogyakarta, Indonesia
The reference of 100% chicken sausage, 100% pork sausage and the mixture of pork-chicken sausages were prepared in the laboratory
Summary
Chicken sausages are one of the commonly consumed meat-based products worldwide. Due to the price difference among raw meat materials, some unethical sellers try to substitute chicken with pork (Kitpipit et al, 2014). Several methods have been developed for the detection of pork adulteration in food products such as meatballs and sausages, namely FTIR spectroscopy combined with multivariate data analysis (Rohman et al, 2011), nuclear magnetic resonance (Nurrulhidayah et al, 2015), the combination of electronic nose and gas chromatographymass spectrometry (Nurjuliana et al, 2011), differential scanning calorimetry (Mansor et al, 2012), twodimensional gas chromatography-mass spectrometry time of flight (Indrasti et al, 2010), and liquid chromatography (Rohman et al, 2012) Some of these methods are lack in sensitivity such as FTIR spectroscopy and complex instrumentation like NMR spectroscopy and 2D chromatography, specific method based on the DNA detection using real-time polymerase reaction has emerged as method of choice
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