基于ARMS-PCR技术检测SLC39A13基因核苷酸多态性方法的建立  

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

作　　者：郭冰茜 李萌钰 王瑞 王树松 冯惠勇[1] 李天明[1] GUO Bingqian;LI Mengyu;WANG Rui;WANG Shusong;FENG Huiyong;LI Tianming(School of Food and Biology,Hebei University of Science and Technology,Shijiazhuang,Hebei 050018,China;Forestry Institute,Northeast Forestry University,Harbin,Heilongjiang 150006,China;Taiyuan Jinyu Clinical Inspection Office Company Limited,Taiyuan,Shanxi 030003,China;Hebei Key Labratory of Reproductive Medicine,Hebei Hospital of Reproductive Health,Shijiazhuang,Hebei 050071,China)

机构地区：[1]河北科技大学食品与生物学院,河北石家庄050018 [2]东北林业大学林学院,黑龙江哈尔滨150006 [3]太原金域临床检验所有限公司,山西太原030003 [4]河北省生殖健康医院河北省生殖医学重点实验室,河北石家庄050071

出　　处：《河北科技大学学报》2024年第5期497-507,共11页Journal of Hebei University of Science and Technology

基　　金：国家科技支撑计划项目(2015BAD15B0501);中央引导地方科技发展资金项目(226Z7722G)。

摘　　要：为了实现核苷酸多态性(SNP)的精准分型,针对SLC39A13基因rs755555位点,建立基于荧光定量PCR的分子诊断技术。首先,分别设计rs755555位点及内参基因peptidylprolyl isomerase A(PPIA)的Taqman荧光ARMS-PCR检测引物和探针;其次,构建阳性对照质粒;最后,以基因分型精确度为指标,优化引物探针组合,以及检测试剂的PCR反应体系和反应条件。结果表明:野生型最优引物探针组合为WF1、R1、FP1、PIRF5、PIRR5、PIRP5,突变型最优引物探针组合为FMF3、R1、FP1、PIRF5、PIRR5、PIRP5;每个检测样品的最优反应体系为SLC39A13基因上下游引物探针各0.1μL,内标上下游引物探针各0.1μL,10μL PerfectStart^(■)ⅡProbe qPCR SuperMix UDG,5.4μL纯水,4μL样品基因组。重复性实验和70个样品的检测验证,确认了检测体系的可行性,为研发SLC39A13基因rs755555位点多态性检测试剂盒提供了技术基础。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:0.1μL each of upstream and downstream primers and probes for the SLC39A13 gene,0.1μL each of upstream and downstream primers and probes for the internal standard,10μL PerfectStart^(■)ⅡProbe qPCR SuperMix UDG,5.4μL purified water,and 4μL 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.

关 键 词：分子生物学 人SLC39A13基因 ARMS-PCR 核苷酸多态性检测 实时荧光定量PCR 

分 类 号：Q789[生物学—分子生物学]