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.