For each primer pair, the annealing temperature described in the literature was initially used (Table 2). When non-specific amplification occurred at these initial temperatures, the annealing temperature was increased by 2°C until specific product amplification was obtained or 60°C was reached. When no amplification occurred, the temperature was decreased by 2°C until amplification was obtained or 45°C was reached. Among the 15 primer pairs, Amb82, Sg-2, Sg-6, Sg-8, and ER-HAJZC, did not require a change in the initial annealing temperature.