Identification of pork in meat products using real-time loop-mediated isothermal amplification

Abstract

In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of <i>cytochrome b</i> (<i>cytb</i>) gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%-10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products.