基于高分辨率熔解曲线的GSTM1基因分型方法的建立与评价

Establishment and evaluation of GSTM1 genotyping based upon high resolution melting curve

目的:采用高分辨率熔解曲线(High Resolution Melting,HRM)法检测GSTM1基因多态性,并评价其准确性,以期为临床抗肿瘤药物与抗结核药物的临床个体化应用提供参考。方法:2019年3月—2019年10月就诊于四川省人民医院等3家医疗机构的94例患者的外周静脉血样本,纳入标准为:年龄为16—88周岁;签署知情同意书;留存血液样本的体检患者。采用离心柱法提取其全血基因组DNA,对提取后的DNA进行浓度及纯度检测,并采用HRM法对产物进行分析;同时使用Stata17软件采取随机种子从GSTM1 null和GSTM1 non-null共2种基因型样本中随机选取33例样本使用焦磷酸测序法进行准确性验证。结果:94例样本DNA浓度范围为10~60 ng·μL^(–1),纯度OD260/280位于1.6~2.0之间。所有样本扩增反应均正常,其中37例患者(39.36%)检测到GSTM1 non-null基因型,57例患者(60.64%)检测到GSTM1 null基因型。随机选取的33例样本使用焦磷酸测序进行验证,17例患者(51.52%)检测到GSTM1 non-null基因型,16例患者(48.48%)GSTM1 null基因型。与焦磷酸测序法结果进行比对,HRM法的结果与之完全相符。结论:该研究建立了HRM方法检测GSTM1的实验体系和方法,用于检测GSTM1基因位点的2种不同基因型,准确性高,为临床肿瘤和结核等患者用药方案的优化提供了一种快速、准确的药物基因检测方法,对于抗肿瘤药物与抗结核药物的临床应用具有重要的指导意义。

OBJECTIVE To detect the polymorphism of GSTM1 by high resolution melting(HRM)method and evaluate its accuracy to provide references for individualized dosing of antineoplastic and anti-tuberculosis drugs.METHODS Peripheral venous blood samples were collected from 94 hospitalized patients at three medical institutions from March 2019 to October 2019,including Sichuan Provincial People's Hospital.The inclusion criteria included an age range of(16-88)years,signing informed consent and receiving physical examination with retained blood samples.Genomic DNA was extracted from whole blood by centrifugal column method and the concentration and purity of extracted DNA were determined.HRM analysis was performed.Additionally,33 samples were randomly selected from both GSTM1 null and GSTM1 non-null genotypes for verification through pyrosequencing by Stata17 software.RESULTS DNA concentration in all 94 samples ranged 10-60 ng·μL^(-1)with a purity ratio of OD260/280 ratio(1.6-2.0).All samples underwent normal amplification reactions.Among them,GSTM1 non-null genotype was detected in 37 cases(39.36%)while GSTM1 null genotype in 57 cases(60.64%).And 33 randomly selected samples were verified by pyrophosphate sequencing.There were 17 cases(51.52%)of GSTM1 non-null genotype and 16 cases(48.48%)of GSTM1 null genotype.As compared with pyrophosphate sequencing,the results of HRM method were completely consistent.CONCLUSION This study has successfully established an experimental system and method for detecting GSTM1 by HRM method,which was used to detect two different genotypes of GSTM1 gene locus with high accuracy.It provided a rapid and accurate drug gene detection method for the optimization of drug regimen for patients with clinical tumors and tuberculosis.Moreover,it holded significant guiding implications for the clinical application of anti-tumor and anti-tuberculosis medications.