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