液相芯片技术快速检测2型糖尿病患者相关降糖药物基因多态性的方法学建立

Development of A Liquid Phase Chip Technology for Simultaneous and Rapid Detecting of the Gene Polymorphisms in Medications with Type 2 Diabetes Mellitus

目的建立一种可同时、快速检测2型糖尿病患者降糖药相关药物基因多态性的液相芯片检测系统。方法根据7个目的基因的rs号,在Genbank中查找其靶位点附近碱基序列(目的基因包括磺脲类受体1,转录因子7类似物2,胰岛素受体底物1,过氧化物酶体增殖物激活受体γ,有机阳离子转运蛋白与多药和有毒化合物排出家族,有机阴离子转运蛋白家族成员1B1等),以PrimerPlex软件设计等位基因特异性引物,Primer6.0软件设计含检测位点的PCR引物,通过多重PCR扩增,等位基因特异性引物延伸(ASPE),MagPlex~Tag微球杂交,液相芯片系统Luminex 200检测荧光信号,确定基因型,优化反应体系并以5份代表性标本进行方法学评价。收集2019年1月~12月东莞市厚街医院新诊2型糖尿病患者血液样本115例,采用建立的系统检测上述7个靶位点,并随机选取25例与测序结果比较。结果7个目标基因经PCR扩增,产物电泳成像后清楚可见7条目标条带,无非特异性条带;经优化ASPE杂交条件,选择退火温度55℃,Biotion~dCTP浓度与dCTP浓度的比值为3∶1,37℃孵育45 min时检测效果最佳;临床样本检测结果显示:纯合子荧光强度中位值(median fluorescence intensity,MFI)比率均>0.9或<0.1,杂合子MFI比率在0.4~0.6之间。纯合子MFI比率批内批间CV在0.9%~3.3%和2.1%~4.6%,而杂合子则在2.9%~7.3%和5.2%~11.2%;DNA最低检测限为0.75ng;25例样本的检测结果与测序结果完全一致,准确度100%。结论该研究成功建立了一种新的液相芯片检测系统,高效便捷,能同时检测7个目的基因型,满足临床的需求。

Objective To establish a method for simultaneous and rapid detecting of the gene polymorphisms in medications,based on liquid phase chip technology.Methods The seven gene sequences near targeted sites related to Sulfonylurea receptor 1,transcription factor 7~like 2,insulin receptor substrate~1,peroxidase proliferation of activated receptor~γ2,organic cation transporters and multidrug and toxin extrusion proteins,Organic Anion Transporters 1B1 were found in Genbank according to their rs number,and the allele specific primers and PCR probes were designed by PrimerPlex software and Primer6.0 software.Through multiple PCR amplification,followed by allele specific primer extension(ASPE),and MagPlex⁃Tag microspheres hybridization,the suspension array Luminex 200 system step⁃by⁃step,the genotypes were determined by fluorescence signal.The reaction system was optimized and its methodological evaluation was performed by five representative specimens.115 patients with type 2 diabetes Mellitus from Dongguan Houjie Hospital were recruited in this study form January 2019 to December.The seven genotypes of the 115 patients were detected by the established method,and the results of 25 patients were compared with the sequencing results.Results Seven target bands were clearly visible after electrophoresis imaging,and no nonspecific bands were found.After optimizing ASPE hybridization conditions,the test results was the best with annealing temperature 55℃,the ratio of Biotion~dCTP concentration to dCTP concentration at 3:1,incubating at 37℃for 45min.The results of 115 samples showed that allelic median fluorescence intensity(MFI)ratios of homozygotes(mutant/wild~type)were all greater than 0.9 or less than 0.1,and all the allelic MFI ratios of heterozygotes were between 0.4 and 0.6.The within run and between run coefficients of variance for homozygotes allelic MFI ratios were 0.9%~3.3%and 2.1%~4.6%,respectively,while 2.9%~7.3%and 5.2%~11.2%,respectively for heterozygotes.The minimum DNA template requirements were 0.75ng.The genotypes of 25 patients determined by the established method were completely concordant with the sequencing results.Conclusion A method based on liquid phase chip technology for simultaneous and rapid detecting of the gene polymorphisms in medications has been successfully developed,which can meet the needs of clinical..