多重STR⁃PCR在异基因造血干细胞移植后嵌合率检测中的应用  

Application of multiple STR⁃PCR in the detection of chimerism rate after allogeneic hematopoietic stem cell transplantation

作　　者：彭智勇 彭瀚慧 肖成 汤浩然 裴艳茹 台运春 PENG Zhiyong;PENG Hanhui;XIAO Cheng;TANG Haoran;PEI Yanru;TAI Yunchun(Department of Hematology,Nanfang-Chunfu Children's Institute of Hematology,Taixin Hospital,Dongguan,Guangdong,China,523125;Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification,School of Forensic Medicine,Southern Medical University,Guangzhou,Guangdong,China,510515;Department of Science and Education,Guangzhou Red Cross Hospital,Guangzhou,Guangdong,China,510220)

机构地区：[1]东莞台心医院&南方春富血液病研究院血液病科,广东东莞523125 [2]广州市法医多组学精准鉴识重点实验室,南方医科大学法医学院,广东广州510515 [3]广州市红十字会医院科教科,广东广州510220

出　　处：《分子诊断与治疗杂志》2024年第12期2256-2260,共5页Journal of Molecular Diagnostics and Therapy

基　　金：国家自然科学基金面上项目(82372312)。

摘　　要：目的探讨GoldeneyeTM 20A试剂盒检测异基因造血干细胞移植后嵌合率的准确性及stutter峰对结果的影响。方法采集18名无亲缘关系健康志愿者的外周血,随机两两配对,按其白细胞比例混合制备出不同供受者细胞比例的多组体外嵌合体模型,用GoldeneyeTM 20A试剂盒对19个STR基因座进行复合扩增,扩增产物进行毛细管电泳,再分别用忽略stutter峰(方法一)和考虑stutter峰(方法二)两种方法计算嵌合率。结果总共63份混合样本均可检出供受者特异性STR位点的等位基因,即最小检出细胞嵌合率可低至0.625%。仅模拟嵌合率为40%的组,两种算法所得结果差异无统计学意义(t=1.046,P>0.05);当模拟嵌合率≤20%时,两种算法所得结果差异有统计学意义(P均<0.05)。用方法一计算时,40%、20%、10%三个模拟嵌合率组的实测值与理论值的比较差异无统计学意义(P均>0.05);模拟嵌合率5%、2.5%、1.25%、0.625%的组,实测值与理论值比较,差异有统计学意义(P均<0.05)。用方法二计算时,40%、20%、10%、5%四个模拟嵌合率组的实测值与理论值的比较,差异无统计学意义(P均>0.05)。模拟嵌合率2.5%、1.25%、0.625%的组,实测值与理论值比较,差异有统计学意义(P均<0.05)。两种算法所得嵌合率实测值与理论值均呈显著直线相关(P<0.05),但方法二的回归直线在Y轴上的截距0.067比方法一1.9852更接近于0。结论用多重STR⁃PCR结合毛细管电泳法检测嵌合率灵敏度高、准确性强,通过选取理想的位点,将stutter峰面积计算在主峰面积之内比忽略stutter峰直接计算的结果更准确。Objective To evaluate the accuracy of quantitative determination of chimerism using GoldeneyeTM20A testing kit,and the impact of stutter peaks on the results.Methods We mixed pairwise blood samples collected from 18 healthy donors to prepare chimera models with different ratios of donorrecipient WBCs.DNA was extracted from the mixed samples,and 20 fluorescence labeled STR loci were amplified using GoldeneyeTM 20A testing kit,and then sequenced by capillary electrophoresis.Finally,the chimerism was calcu⁃lated in the case of considering(method 2)and ignoring stutter peaks(method 1).Results A total of 63 mixed samples were able to detect alleles of donor recipient specific STR loci,with a minimum detectable cell chimerism rate as low as 0.625%.Only the group with a simulated chimerism rate of 40%showed no statistically significant difference in the results obtained by the two algorithms(P>0.05).When the simulated chimerism rate was≤20%,there was a statistically significant difference in the results obtained by the two algorithms(P<0.05).When using Method 1 for calculation,there was no statistically significant difference between the measured values and theo⁃retical values of the 40%,20%,and 10%simulated chimeric rate groups(P>0.05).The groups with simulated chi⁃merism rates of 5%,2.5%,1.25%,and 0.625%showed statistically significant differences between the measured values and theoretical values(P<0.05).When using Method 2 for calculation,there was no statistically significant difference between the measured values and theoretical values of the four simulated chimeric rate groups of 40%,20%,10%,and 5%(P>0.05).The groups with simulated chimerism rates of 2.5%,1.25%,and 0.625%showed statistically significant differences between the measured values and theoretical values(P<0.05).The measured values of the embedding rate obtained by both algorithms show a significant linear correlation with the theoretical values(P<0.05),but the intercept of the regression line in method two on the Y⁃axis is 0.067,which is clo

关 键 词：造血干细胞移植 短串联重复序列 嵌合率 stutter峰 

分 类 号：R457.7[医药卫生—治疗学]