2.2.4 Design and synthesis of primers and probes
For the single-tube monochromatic fluorescence method, according to the technical principle of ARMS-PCR, two specific upstream primers, one common probe, and one common downstream primer were designed using Primer 5.0 software. The probe was labeled with a FAM fluorescent dye at the 5′-end and with a BHQ1 fluorescence quenching group at the 3′-end. The primer probe sequence is shown in Supplement 2. For the single-tube two-color fluorescence method, in which one tube was used to detect the mutation of two sites simultaneously, the detection probe of one site was labeled with a FAM fluorescent dye at the 5′-end, the detection probe of the other site was labeled with a VIC or HEX fluorescent dye, and the 3′-end was labeled with a BHQ1 fluorescence quenching group; the gene sequences are shown in Supplement 3.