3.5 Verification of the ARMS-PCR combined with fluorescent probe technology system
According to the PAH mutations in Shaanxi province and the mutations of the PAH gene in China, we selected 9 mutation sites covering greater than 50% of the mutations of the PAH gene in Shaanxi province: c.728G>A (p.R243Q), c.1197A>T (p.V399V), c.331C>T (p.R111X), c.1068C>A (p.Y356X), c.611A>G (EX6-96A>G), c.1238G>C (p.R413P), c.721C>T (p.R241C), c.442-1G>A (IVS4-1G>A), and c.158G>A (p.R53H). We used ARMS-PCR combined with fluorescent probe technology to design and develop a kit to detect common mutations of the PAH gene. Refer to Supplement 6 for a comparison between the results of the monochromatic fluorescence method and NGS. After exploring the experimental conditions, optimizing the experimental procedures, and calibrating the temperature control system of the instrument, 117 samples from 40 families were used to verify the performance of the ARMS-PCR combined with fluorescent probe technology system, and the results were compared with those from NGS (Table 3). Taking NGS as the gold standard, the detection sensitivity of ARMS-PCR combined with fluorescent probe technology was 94.17% (97/103). Fifty-three samples without a mutation as determined by NGS were also found to be negative with the newly developed approach, indicating that the detection specificity of the ARMS-PCR combined with fluorescent probe technology system can reach 100%.
Six mutation sites were not detected by ARMS-PCR combined with fluorescent probe technology (twice each for c.158G>A, c.331C>T, and c.442-1G>A) which was inconsistent with the results of NGS. Factors, such as experimental operation, instrument stability, and repeated freezing and thawing of the reagents, were excluded as contributing to the lack of detection. As such, we considered that the sensitivity of the detection reagents needs to be improved.
Table 3 Comparison of PCR detection and sequencing results for 117 individuals from 40 families