荧光定量PCR染料法在HLA-B*5801等位基因检测中的性能评价及应用  

Performance evaluation and application of fluorescent quantitative PCR dye method in HLA-B*5801 allele detection

作　　者：梁海[1] 夏茹楠 赵欢欢 张鹏程 鲁宁 林紫薇[1] 吴炜[1] LIANG Hai;XIA Ru-nan;ZHAO Huan-huan;ZHANG Peng-cheng;LU Ning;LIN Zi-wei;WU Wei(Department of Pharmacy,People's Hospital of Bozhou,Bozhou 236800,China)

机构地区：[1]亳州市人民医院药学部,亳州236800

出　　处：《药物分析杂志》2022年第12期2092-2100,共9页Chinese Journal of Pharmaceutical Analysis

基　　金：安徽省重点研究与开发计划项目(1804h08020296);亳州市重点研发计划项目(bzzc2021022);亳州市人民医院科研孵化项目(Byf202005)。

摘　　要：目的:通过检测评估荧光定量聚合酶链反应(PCR)染料法对人类白细胞抗原B位点5801(HLA-B*5801)等位基因的准确性、灵敏度及其在干扰因素下的结果分析研究,同时与PCR-SBT测序法进行对比。方法:收集45例痛风、高尿酸血症患者和20例正常人群样本,利用HLA-B*5801等位基因检测试剂盒进行检测,同时利用测序法对HLA-B等位基因高度突变的2/3/4外显子正反测序。灵敏度验证研究,随机选取1例阳性样本,按照HLA-B*5801等位基因检测试剂盒说明书要求稀释至最低浓度,检测结果与测序结果对比;抗干扰能力验证研究,随机选取3例阳性样本,加入规定要求的胆红素、脂质、阿司匹林作为干扰因素,利用PCR染料法检测,检测结果与测序结果对比。结果:45例痛风、高尿酸患者中有8例HLA-B*5801基因阳性,阳性率为17.8%;20例正常人群则有1例携带HLA-B*5801基因,阳性率5%。最低浓度样本也可正确检出结果,PCR染料法的准确性、灵敏性、特异性均为100%;3例阳性患者在胆红素、脂质等干扰物质影响下仍可正常检出,抗干扰能力良好。结论:PCR染料法可正确检测HLA-B*5801基因,灵敏度较好且在干扰物质存在的状况下结果不受影响,与测序结果一致,且操作更快捷方便。Objective:To evaluate the accuracy and sensitivity of fluorescent quantitative polymerase chain reaction(PCR)dye method for detecting the allele of human leukocyte antigen B locus 5801(HLA-B*5801)and analyze the results under interference factors,and to compare it with PCR-SBT sequencing method.Methods:45 patients with gout,hyperuricemia and 20 normal people were collected and tested with HLA-B*5801 allele detection kit,while 2/3/4 exons with highly mutated HLA-B allele were sequenced by sequencing.In the sensitivity validation study,one positive sample was randomly selected and diluted to the lowestconcentration according to the instructions of HLA-B*5801 allele detection kit,and the test results were compared with the sequencing results;In the anti-interference verification study,three positive samples were randomly selected,bilirubin,lipid and aspirin were added as interference factors,PCR dye method was used for detection.The test results were aligned to the sequencing results.Results:HLA-B*5801 gene was positive in 8 of 45 patients with gout and hyperuricemia,the positive rate was 17.8%;One of the 20 normal people carried HLA-B*5801 gene,with a positive rate of 5%.The accuracy,sensitivity and specificity of PCR dye method are all 100%;Three positive patients could still be detected normally under the influence of bilirubin,lipid and other interfering substances,and their anti-interference ability was good.Conclusion:The PCR dye method can correctly detect the HLA-B*5801 gene,with good sensitivity and unaffected results in the presence of interfering substances,consistent with the sequencing results,and faster and convenient to operate.

关 键 词：PCR染料法 HLA-B*5801 准确性 灵敏度 抗干扰能力 

分 类 号：R917[医药卫生—药物分析学]