实时荧光定量PCR在HLA-B~*5801等位基因检测中的性能评价  

Performance evaluation of real-time fluorescence quantitative PCR in the determination of allele HLA-B~*5801

作　　者：吴洁[1] 伊洁[1] 甘勇[1] 徐英春[1] WU Jie;YI Jie;GAN Yong(Department of Clinical Laboratory,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]中国医学科学院北京协和医院检验科

出　　处：《中国医学装备》2020年第2期30-33,共4页China Medical Equipment

摘　　要：目的:评价采用实时荧光定量聚合酶链反应(PCR)方法检测人类白细胞抗原B位点5801(HLA-B*5801)等位基因的重复性、准确性和最低检出限。方法:选取HLA-B*5801阳性和阴性样本各2例,每一样本每批内测定5次,连续测定2 d,共重复测定10次,评价方法的重复性。收集20例已知阳性和阴性样本,实时荧光定量PCR方法进行检测,同时与金标准DNA测序法的结果进行比对,评价方法的准确性。同时进一步选取其中1例阳性DNA样本进行梯度稀释后再行检测,评价方法的最低检出限。结果:4例样本重复性检测阳性和阴性符合率为100%;实时荧光定量PCR法与DNA测序法检测结果符合率为100%,收集的20例已知阳性和阴性样本中HLA-B*5801阳性8例,阴性12例;实时荧光定量PCR的最低检出限为2.5 ng/μl DNA。结论:实时荧光定量PCR可方便、快捷地检测HLA-B*5801等位基因,其准确度高,重复性好,适用于临床常规样本的检测。Objective:To evaluate the repeatability,accuracy and limit of detection of real-time fluorescence quantitative polymerase chain reaction(PCR)in detecting HLA-B*5801.Methods:Two positive samples of HLA-B*5801 and two negative samples of HLA-B*5801 were selected,and each sample was measured 5 times in each batch.The measurement was repeated in 2 day,and each sample was repeatedly tested 10 times to evaluate the repeatability of this method.20 known HLA-B*5801 positive and negative samples were collected and were detected by real-time fluorescence quantitative PCR.The results were compared with the results of DNA sequencing that was the gold standard method so as to evaluate the accuracy of this method.Furthermore,one of the positive DNA samples was selected for gradient dilution,and then the detected results were used to evaluate the limit of detection.Results:The results of real-time fluorescence quantitative PCR showed the positive and negative coincidence rate of repeatability test of 4 samples was 100%.The coincidence rate between real-time fluorescence quantitative PCR and DNA sequencing was 100%.In 20 known positive and negative samples,8 cases were positive for HLA-B*5801 and 12 cases were negative for HLA-B*5801.The limit of detection of real-time fluorescence quantitative PCR was 2.5 ng/μL DNA.Conclusion:Real-time fluorescent quantitative PCR can detect allele HLA-B*5801 easily and quickly,and it has high accuracy and good repeatability.It is suitable for the detection of clinical routine samples.

关 键 词：实时荧光定量 聚合酶链反应(PCR) 类白细胞抗原B位点5801 性能评价 

分 类 号：R197.39[医药卫生—卫生事业管理]