Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop

•Novel primers yielded a highly pig-specific PCR product of 712bp.•PCR assay could detect pork tissue in raw, cooked and adulterated samples.•PCR signal (712bp) was observed even in autoclaved pork samples (121°C).•PCR assay is able to detect pork adulteration up to the extent of 0.1%.•Limit of detection (LOD) of pig DNA is as low as 10pg (pico grams). We describe a highly specific PCR assay for the authentic identification of pork. Accurate detection of tissues derived from pig (Sus scrofa) was accomplished by using newly designed primers targeting porcine mitochondrial displacement (D-loop) region that yielded an unique amplicon of 712 base pairs (bp). Possibility of cross-amplification was precluded by testing as many as 24 animal species (mammals, birds, rodent and fish). Suitability of PCR assay was confirmed in raw (n=20), cooked (60, 80 and 100°C), autoclaved (121°C) and micro-oven processed pork. Sensitivity of detection of pork in other species meat using unique pig-specific PCR was established to be at 0.1%; limit of detection (LOD) of pig DNA was 10pg (pico grams). The technique can be used for the authentication of raw, processed and adulterated pork and products under the circumstances of food adulteration related disputes or forensic detection of origin of pig species.