A multiplex real-time PCR method for differentiation of beef, buffalo meat and pork

Abstract

The objective of this study was to optimize a multiplex real-time PCR protocol for detection of DNA of beef, buffalo meat and pork, serving for food authenticity. The optimized concentrations were 200 nM primer and 100 nM specific probe for beef/buffalo meat, and 300 nM primer and 150 nM probe for pork. The amplification was performed using initial denaturation at 50oC for 2 min, 95oC for 2 min, followed by 45 cycles of denaturation at 95oC for 15 sec, and annealing and extension at 60oC for 40 sec. This protocol had high sensitivity and specificity. The detection limit of this method was found to be 0.1% in raw and heat-treated meat mix (80 – 121oC/15 min) or 0.005 ng DNA/reaction. The protocol of testing was applied for the commercial products both fresh and processed meats. The results demonstrated that 50% of raw beef sample (6/12) weren't found beef DNA. Eight of twelve beef sausage samples (66.67%) contained buffalo DNA. Beef DNA were found in all 12 samples of beef meatballs, but eight out of the 12 meatball samples were confirmed to have buffalo DNA (66.67%) and two out of the 12 meatball samples (16.67%) also contained porcine DNA.