Establishment of the detection system
We used the wild-type CYP2C19 * 2/3/17 plasmid and mutant CYP2C19 *
2/3/17 plasmid as real samples to simulate wild-type and mutant CYP2C19
* 2/3/17 genomic sequences, respectively. A primer probe sequence for
CYP2C19 * 2/3/17 type was designed, as shown in Table S2; CYP2C19 * 3
was designed in the form of a Taqman probe, and CYP2C19 * 2 and CYP2C19
* 17 were designed in the form of two adjacent probes. PCR amplification
was performed by asymmetric amplification of the upstream and downstream
primers. Finally, the optimal detection reaction system was determined,
and the detection time was within 1 h. The established reaction system
accurately identified and distinguished different sites and genotypes:
CYP2C19 * 2 wild-type and mutant (Figure 2A), CYP2C19 * 3 wild-type and
mutant (Figure 2C), CYP2C19 * 17 wild-type and mutant (Figure 2E),
CYP2C19 * 2/3 heterozygous (Figure 2B), CYP2C19 * 2/17 heterozygous
(Figure 2D), CYP2C19 * 3/17 heterozygous (Figure 2F) and CYP2C19 *
2/3/17 heterozygous (Figure 2G). Based on the experimental results, the
system can accurately distinguish different sites and genotypes. Then,
we designed sequencing primers, as shown in Table S2, and polymorphic
sites were amplified by PCR and sent for Sanger sequencing. The
sequencing results are shown in Figure 3: wild-type, mutant and
heterozygous CYP2C19 * 2, respectively, in Figure 3A-C; wild-type,
mutant and heterozygous CYP2C19 * 3, respectively, in Figure 3D-F; and
wild-type, mutant and heterozygous CYP2C19 * 17, respectively, in Figure
3G-I. The sequencing results for the different genotypes of CYP2C19 *
2/3/17 were consistent with those of RPCR-MC.