So was obtained by diluting 100-fold the DNA standard, then S0 was diluted by a constant 10-fold gradient to prepare S1, S2, S3, S4, S5, S6 and S7 samples, which were served as standard curve samples. The reaction system was constituted by the modified DNA template 3uL, the upstream1uL, the downstream primers 1uL, the methylated probe 1uL, the unmethylated probe 1uL, 2 × Goldstar TaqMan mixture 15 uL, and RNase-Free Water added to 30 uL. The reaction conditions were as follows: denaturation at 95 °C for 10min, denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s and circulation 40 times. The samples were repeated 3 times and the mean values were accepted. After part of the PCR products were connected to plasmids that were serving as carriers, they were sequenced by universal primers. The results showed that the sequences of the target sequences were consistent with the sequences designed by Beacon Designer software.
