Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products

Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3' end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.