The RPCR-MC method
Each RPCR-MC analysis reaction mixture contained 1.25 µl 5 U/µl D-Taq enzyme, 1 µl dNTP Mix (10 mM), 5 µl 5XPCR Buffer, 0.3 µl 2C19 * 3-F1 (10 µM), 1.2 µl 2C19 * 3-R2 (100 µM), 1.2 µl 2C19 * 3-P5 (100 µM), 0.25 µl 2C19 * 17-F1 (5 µM), 0.1 µl 2C19 * 17-R1 (50 µM). 0.1 µl 2C19 * 17-P1 (50 µM), 0.1 µl 2C19 * 17-P2 (50 µM), 0.3 µl 2C19 * 2-F2 (10 µM), 0.12 µl 2C19 * 2-R1 (100 µM), 0.12 µl 2C19 * 2-P1 (100 µM), 0.12 µl 2C19 * 2-P2 (100 µM) mixture, 9.84 µl deionized water and 4 µl DNA template. 2C19 * 3 was designed as a TaqMan probe, whereas 2C19 * 2 and 2C19 * 17 were designed as adjacent probes. The PCR and melting curve analysis were performed using a SLAN-96S (Shanghai Hongshi) PCR instrument. The PCR involved denaturation at 95°C for 30 s, 45 cycles of 95°C for 2 s and 60°C for 5 s, followed by 72°C for 1 min,90°C for 1 min,37°C for 1 min, melting curve analysis was from 45°C to 80°C for 0.06 °C/s.