2xES Taq Master Mix reagent instructions were followed. The reaction system was constituted by the modified DNA template 1 uL, the upstream primer 1 uL, the downstream primers 1uL, 2x ES Taq Mix 25 uL, and ddH2O 20 uL. The reaction conditions were as follows: denaturation at 94°C for 4 min, denaturation at 94°C for 15 s, annealing at 56°C for 30 s, extension at 72°C for 10 s, extension at 72°C for 7 min, and 31 cycles. The 2% agarose gel was prepared by electrophoresis to detect whether the PCR products contained the target fragments (111bp). The target fragments were recovered and dissolved, and the solution of DNA was collected. The concentration of DNA recovered was measured with a spectrophotometer, and the DNA sample was used as DNA standard. The OD value (260 nm) of CHN2 was 0.05 with a concentration of 210 ng/uL, and the Copies value was 2.1E + 12 Copies/uL. DNA Copies/uL = (mass/molecular weight) × NA = NA × [DNA concentration (ng/uL) × 1uL] × 10-9/(DNA molecular weight × 660).
