Discrimination of two alleles of the melanocortin receptor 1 gene to discern European wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) in meat products by real-time PCR

Abstract

A duplex real-time polymerase chain reaction (PCR) technique for the discrimination of two subspecies of sus scrofa in meat products has been developed. Primers and probes target at a sequence in the melanocortin receptor 1 (MCR1) gene being associated in the expression of wild-type coat color. Both PCRs amplify a 56-bp product of DNA from wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) likewise. One of the TaqMan probes specifically anneals to the wild boar sequence and the other one to the domestic pig sequence, their base sequence differing only in a single nucleotide (single nucleotide polymorphism, SNP). This qualitative-method allows the detection of genomic DNA from wild boar and domestic pig as low as 25 pg in a 25-μl reaction volume. No cross reactivities were found with either genomic DNA from various other meat species, or with other ingredients of meat products (e.g. spices). The PCR efficiency is >95% for both targets. Although both PCRs are impaired by the presence of bovine and porcine DNA (wild boar detection is impaired by domestic pig DNA and vice versa), the method is applicable for the detection of low levels of wild boar and domestic pig meat simultaneously in commercial meat products. The limit of detection (LOD) in meat samples is 2% for wild boar and 5% for domestic pig.