Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C (q) (quantification cycle) from 10 ng/mu l porcine DNA is (18.70 +/- 0.12 to 19.08 +/- 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r (2) = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork-beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.