飞行时间质谱多基因检测系统检测心血管用药11个基因的性能验证研究

目的对飞行时间质谱多基因检测平台的检测性能进行验证,建立基于飞行时间质谱多基因检测平台的性能验证研究方案。方法选择2017年9月至2018年10月来自中国医学科学院阜外医院已测部分心血管用药相关基因位点的DNA标本998例和全血标本20例,其中男性512例,女性506例。18~30岁280例,31~64岁442例,≥65岁296例。用飞行时间质谱法对998份DNA标本心血管用药相关11个基因进行检测,检测结果与已知结果进行比对,评价998例已测标本符合率;选择20例全血标本,对心血管用药相关11个基因分别进行飞行时间质谱检测和Sanger测序,对比两次结果,以Sanger测序为金标准进行准确度评价。从998例DNA标本中选择部分样本进行以下研究,选择10份包含所有位点为野生型的基因组DNA,用飞行时间质谱检测,进行特异性评价;选择4份包含所有位点杂合基因型标本,梯度稀释后检测,评价位点的测定下限;选择14份包含中国人群所有常见位点基因型标本,进行批间和批内精密度评价;选择2份包含代表性峰形的野生和纯合突变标本各1份,进行抗干扰研究。结果该方法单次检测的符合率高于99.5%,飞行时间质谱平台与Sanger测序法检测完全一致,测定下限为0.4 ngDNA,批间和批内精密度均为100%,UNG酶的降解能力为10^5拷贝/μl的气溶胶,反应体系对基因组和PCR各中间产物气溶胶有很强的抗干扰能力,点样及检测过程中芯片不同基质间无交叉污染。结论飞行时间质谱多基因检测系统检测性能良好,可应用于临床检测,并且本文建立了基于飞行时间质谱平台多基因检测系统的性能验证研究方案,为广大科研和检验工作者研究该平台打下基础。

Performance verification of a time-of-flight mass spectrometry based cardiovascular drug-related polygene detection system

Objective Toestablish and verify the method of genetic polymorphisms using time-of-flight mass spectrometry as a polygene testing platform.Methods 998 cases of DNA samples and 20 cases of whole blood samples were collected from Fuwai hospital of Chinese academy of medical sciencesduring September 2017 to October 2018,including 512 cases of males and 506 cases of females.280 patients aged 18-30,442 patients aged 31-64,and 296 patients aged≥65.11 cardiovascular drugsrelatedgenes in 998 DNA samples were detected by time-of-flight mass spectrometry to evaluate the compliance rate compared with identifiedresults.20whole blood samples were selected to detect 11 genes using both time-of-flight mass spectrometry and Sanger sequencing.The results were compared twice,and accuracy was evaluated according to Sanger sequencing as the gold standard.Ten cases of genomic DNA with wild-type loci were selected for specific evaluation by time-of-flight mass spectrometry.Samples containing all heterozygous genotypes were measured after gradient dilution to evaluate the detection sensitivity of the new method.Samples containing all 49 genotypes(two genotypes were not found because they are rare in Chinese population)were used in order to do the inter-assay and intra-assay precision evaluation.An anti-interference study was performed by selecting wild and homozygous mutant samples of represented heterozygous peak shape.Results The results showed that the compliancerate of the single retrospective sample was over 99.5%.The resultsof time-of-flight mass spectrometry and Sanger sequencing was the same.The minimum detection limit of DNA was 0.4 ng,the inter-assay and intra-assay precision were 100%,and the degradation ability of the UNG enzyme was 10^5 copies/μl in aerosol.The reaction system has a strong anti-interference ability to the genome and intermediate aerosol,and no cross-contamination between different matrices of the chip.Conclusions The time-of-flight mass spectrometry as a polygene detection system shows agood detection performance and can be applied to clinical detection.In addition,this paper established a performance verification research scheme based on the time-of-flight mass spectrometry platform polygene detection system.