3.6 Verification of the two-color fluorescence method
The results of ARMS-PCR combined with fluorescent probe technology were compared with the results of NGS(Supplement 7). We found that when DNA extracted from dried blood spots was used as the template and the concentration was approximately 3 ng/μL, the kit could detect the mutation sites of the positive samples with the two-color fluorescence method, the ΔCT value was less than 35, and the wild-type samples were negative. At the same time, the detection results were consistent with the known positive samples. When DNA extracted from whole blood was used as the template and the concentration was 100–200 ng/μL, using the two-color fluorescence method, the kit could detect the mutation sites of each positive sample, the ΔCT value was less than 32, and the wild-type samples were negative. At the same time, the detection results of the new approach were consistent with the known positive sample gene mutation sites (Supplement 8). The kit was used to analyze whole blood and dried blood spot samples from 126 cases confirmed by sequencing as PKU. After DNA extraction and PCR amplification, ARMS-PCR combined with fluorescent probe technology could accurately detect the positive samples, and the positive mutation sites detected by the kit were exactly the same as those detected by NGS (Table 4).
Table 4 Summary of PCR and sequencing results for 126 clinical samples