Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be $85.56{\pm}0.07^{\circ}C$ for cattle, $84.96{\pm}0.08^{\circ}C$ for pig, and $85.99{\pm}0.05^{\circ}C$ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); $84.91{\pm}0.11^{\circ}C$ for goat and $83.90{\pm}0.11^{\circ}C$ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and $86.31{\pm}0.23^{\circ}C$ for chicken, $88.66{\pm}0.12^{\circ}C$ for duck, and $84.49{\pm}0.08^{\circ}C$ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from $10pg/{\mu}L$ to $100fg/{\mu}L$ levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.