The sequences of the primers were designed to form a mismatch at the base(s) adjacent to the SNP site in the forward primers. Using a BIORAD T-100 thermal cycler (BIO-RAD, CA, USA), the specificity of the primers was assessed empirically. The lengths of the primers and/or the number of mismatched bases within two bases were changed, and finally, the sequences of five pairs of primers and the optimized annealing temperatures were determined