Fast and Sensitive Real-time PCR-based Detection of Porcine DNA in Food Samples by Using EvaGreen Dye

Accidental and/or incidental adulterations of foods by porcine ingredients are common in the globalized food processing industry. Food mislabelling and fraudulent substitutions of non-porcine ingredients with porcine ones are objectionable to those who abstain from porcine derived products due to habitual (e.g., vegans and vegetarians), medical (e.g., porcine allergies), legal (fraudulent labelling), economic (e.g., substitution of expensive meat with cheaper pork meat) and cultural or religious grounds (e.g., Islamic and Jewish dietary restrictions). Thus, a strong demand exists for a fast and sensitive method for quantitative sensing of porcine DNA in food. In this study, we are reporting the development of probe-free real-time PCR assay with new primer sets targeting the cytochrome b gene for the fast and sensitive detection of porcine DNA in real food samples. Standard curve was developed with six ten-fold dilutions of the DNA standard and the assay successfully detected up to 0.00001 ng/mu L of porcine DNA and as low as 0.001 % porcine adulteration in raw pork-chicken binary mixture. The standard curve indicated a linear regression of R-2 value of 0.990 and an efficiency of 92.5 %. The Ct value range for the detected pork DNA from the 35 food samples tested was 16.03-28.76. We confirmed the assay's specificity to porcine DNA against nine non-porcine animal species and 6 vegetables species.