Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction

A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109bp fragment of porcine cytochrome b gene. Specificity test with 10ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5±0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100–0.01% contaminated pork with 102% PCR efficiency, high linear regression (r2=0.994) and ≤6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89±0.16 and 16.37±0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision. ► A real-time PCR assay suitable to determine pork adulteration in meatballs was developed. ► The method combined the uses of species-specific primers and TaqMan probe. ► A short (109-bp) fragment of cytochrome b gene was used as porcine targets. ► The assay was optimized in a complex background, considering all potential factors. ► The assay quantified 0.01–100% pork adulterant in beef meatballs with ≤6% errors.