基于单核苷酸多态性分型的系列特异嵌合体定量方法的建立

Quantitative analysis of lineage-specific chimerism using real-time PCR and single nucleotide polymorphisms

目的建立基于单核苷酸多态性（SNP）分型的系列特异嵌合体定量方法。方法用免疫磁珠分选技术分离CD3＋T淋巴细胞和CD？s粒细胞，利用TaqMan探针建立实时荧光定量聚合酶链反应（PCR）法，对模拟血细胞嵌合体进行检测，并作出标准曲线，以评价该法的灵敏性和特异性。结果免疫磁珠分选技术分选血细胞纯度可达94％～97％。所选的SNP位点在供受者间至少有两个可区分彼此的信息位点。扩增产物中供受者嵌合百分比的对数同实时荧光定量PCR检测的Ct值显著直线相关，线性相关系数r〉0．98，灵敏度不低于0．1％，特异性亦较好。采用实时荧光定量SNP—PCR定量分析同期4例临床病例，根据模拟嵌合体标准曲线对其中1例计算，得到的嵌合率为87．50％。结论基于SNP分型的TaqMan探针实时荧光定量PCR法灵敏度高，特异性好，可用于造血干细胞移植术后血细胞嵌合体的检测。

Objective To develop a real-time quantitative PCR method with TaqMan probe to analysis the lineage-specific chimerism based on single nucleotide polymorphisms （SNP）. Methods CD3 positive and CD15 positive cells were separated by magnetic cell sorting system from cord blood, and a quantitative method were established using real-time quantitative PCR and SNP. Detect the artificial chimerism and mark the standard curves. The reaction system was optimized, and the sensitivity and specificity were evaluated. Results Separation purity of blood cells by magnetic cell sorting system was up to 94 %-97 %. Discrimination between donor and recipient was possible. Dilution experiments of the mock chimerism sample revealed that Ct values correlated linearly with the logarithm of recipient/donor DNA fraction （r 〉 0.98）, and the limit of detection for a minor DNA percentage was under 0.1%, and the specificity was also good.Quantitative analysis of 4 clinical cases in same period were made by real-time fluorescence quantitative SNP-PCR, the fitted rate was 87.50 % based on the chimera standard curve calculated for 1 case. Conclusion The method shows high sensitivity and specificity, and will be used to quantify the lineage- specific chimerism after allogeneic hematopoietic stem cell transplantation.