Detection of plasmid sensitivity
After distinguishing multiple sites of CYP2C19 * 2/3/17 in one tube, we detected the sensitivity of heterozygous samples. First, we prepared CYP2C19 * 2, CYP2C19 * 3, CYP2C19 * 17, and CYP2C19 * 2/3/17 heterozygous samples and diluted them from 106 copies/µl to 100 copies/µl in a 10-fold dilution of 1X TE solution. As depicted in Figure 4A, the detection limit of the CYP2C19 * 2/3 heterozygous sample was a single copy. Similarly, a single copy was detected for CP2C19 * 3 heterozygous samples, CP2C19 * 17 heterozygous samples, and CP2C19 * 2/3/17 heterozygous samples (Figure 4B-D). Furthermore, we tested the CYP2C19 * 2/3/17 heterozygous plasmids at 10 copies/µl and 1 copy/µl in a tube to determine the minimum detection limit, as shown in Figure 4E and F. The unique melting temperature Tm value was obtained as shown in Figure 5. The CYP2C19 * 2/3/17 hybrid plasmid with 10 copies/µl and 1 copy/µl concentrations was repeatedly detected 9 times, and the average TM and standard deviation (SD) of each site were obtained. As indicated in Table S3, the Tm value of the CYP2C19 * 2 mutant was 5.6°C higher than that of wild-type, and the Tm value of the CYP2C19 * 3 mutant was 4.1°C lower than that of wild-type. The Tm value of the CYP2C19 * 17 mutant plasmid was 8.6°C lower than that of wild-type. The results showed that the detection limit of RPCR-MC is 1 copy/µl (or 4 copies per reaction).