Establishment of a method for detecting nucleotide polymorphism of SLC39A13 gene based on ARMS-PCR

In order to achieve its nucleotide polymorphism (SNP) accurate typing, this paper establishes a molecular diagnostic technique based on fluorescent quantitative PCR for accurate genotyping of the nucleotide polymorphism (SNP) at the rs755555 locus of the SLC39A13 gene. Firstly, Taqman fluorescent ARMS-PCR detection primers and probes were designed for the rs755555 locus and the internal reference gene peptidylprolyl isomerase A (PPIA). Secondly, positive control plasmids were constructed. Finally, based on genotyping accuracy, the primer and probe combinations were optimized, and the PCR reaction system and conditions for the detection reagents were optimized. The optimal primer and probe combination for the wild-type was: WF1, R1, FP1, PIRF5, PIRR5, PIRP5; the optimal combination for the mutant type was: FMF3, R1, FP1, PIRF5, PIRR5, PIRP5. The optimal reaction system for detecting samples was: 0.1 μL each of upstream and downstream primers and probes for the SLC39A13 gene, 0.1 μL each of upstream and downstream primers and probes for the internal standard, 10 μL PerfectStart Ⅱ Probe qPCR SuperMix UDG, 5.4 μL purified water, and 4 μL sample genome. The feasibility of this detection system was confirmed through reproducibility experiments and the detection of 70 samples. This provides a technical foundation for the development of a detection kit for the polymorphism at the rs755555 locus of the SLC39A13 gene.