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