A PCR Assay for Identification of Buffalo Origin of Tissue by Amplification of the mt. D-loop Gene

The present study was carried out with aim to develop and standardize the protocol for species-specific PCR assay for detection of tissue of buffalo origin. Muscle tissue samples from viz: cattle (postmortem), buffalo, sheep, goat and pig were used to extract the DNA and the good quality DNA samples having OD260: 280 of 1.8–2.1 were used in this study. Species-specific primers for buffalo was designed through homology comparisons of the mitochondrial gene regions from these species using Megalign (DNA-STAR) and designed primer pairs were tested for their specificity by BLAST analysis. The PCR conditions were optimized in terms quantity and concentration of various components for PCR mix and annealing temperature. The developed assay was evaluated for its species specificity and sensitivity. Efficacy and reliability of developed assay was also validated on known samples, samples from meat admixture and samples subjected to diverse heat treatment viz: boiling, autoclave and microwave. The developed species-specific PCR assay resulted in amplification of DNA template exclusively from buffalo samples and resulted in amplified PCR product of 742bp. Sensitivity of the assay was determined by making 10-fold serial dilution of genomic DNA, which showed that only 10ng of absolute DNA content, was required for PCR amplification and successful identification of tissue of buffalo origin. Thus, it was concluded that developed species-specific PCR assay is effective in identification of tissue of buffalo origin.