TaqMan probe real-time polymerase chain reaction targeting the ATPase 6 gene for the detection of pork adulteration in meat and meatballs

We developed an assay for detecting pork adulteration in meat and meatballs using real-time polymerase chain reaction involving specific primers and a TaqMan probe targeting the porcine mitochondrial (mt) ATPase 6 gene. We proved the specificity of the probe by showing no amplification from DNA isolated from six different meat-providing species: cattle, dog, mouse, chicken, goat, and horse. On the contrary, DNA isolated from pork was positive for amplification, with a Ct (threshold cycle) of 18.69 using a standard amount of DNA template (50ng). The presence of matrix and food processing steps in meatball sample had no influence on the specificity of the probe. The developed technique also has a good repeatability (CV, coefficient of variation = 3.86% for meat and 5.07% for meatballs), showing good linearity and sensitivity, with a limit of detection up to 5 pg of pork total DNA, which equivalent to approximately 6.8 copies of pork mtDNA. In addition, the analysis of spiked pork in beef meatballs showed that the method could determine up to 1% pork contamination. Moreover, the system was successfully applied to detect pork adulteration in commercial meatballs by detecting the presence of pork DNA in two samples.