STR-PCR定量检测嵌合体供者嵌合率准确性探讨

目的探讨用荧光标记复合扩增短串联重复序列(STR-PCR)结合序列分析仪毛细管电泳法检测嵌合体供者细胞嵌合率的准确性。方法采集健康献血者外周血按白细胞比例两两混合制备不同供受者比例的体外嵌合体模型,对15个STR位点进行荧光标记复合扩增,扩增产物进行序列分析仪毛细管电泳,计算嵌合率。结果实验测得嵌合率与扩增前样本嵌合率呈显著直线相关,r=0.999 7,最小检出供者细胞嵌合率<1%。供者嵌合率≥5%的嵌合体模型嵌合率的检测值与理论值差别无统计学意义(P>0.05),<5%的模型嵌合率检测值与理论值差别有统计学意义(P<0.05)。结论荧光标记复合扩增STR基因结合序列分析仪毛细管电泳检测嵌合率方法个体识别力高、特异、敏感,当供者嵌合率≥5%时能准确反映异基因造血干细胞移植后患者的嵌合状态。

Evaluation of the accuracy of STR-PCR to quantitate chimera

Objective To evaluate the accuracy of quantitative multiple short tandem repeat (STR) amplification by fluorescence labeling polymerase chain reaction ( PCR) combined with capillary electrophoresis for the quantitative determination of chimerism. Methods We mixed every two blood samples collected from healthy blood donors to prepare chimera models with different ratios of donor-recipient WBCs. DNA was extracted from the mixed samples, and 15 fluorescence labeled STR loci were amplified, and then sequenced by capillary electrophoresis, and then the engomphosis rates were calculated. Results A linear correlation between the STR-PCR determined chimera rate and the rate of the WBC mixture was found(r = 0.9997) , and the lowest detectable engomphosis rate was < 1%. No statistical difference in chimerism rates between our test results and the theoretical value was found in mixtures with chimerism rates ≥5% (P >0.05) ; however, statistical difference between them was found in mixtures with chimerism rate <5%(P<0.05). Conclusion The chimerism of patients after allogeneic stem cell transplantation can be accurately determined by STR-PCR when the chimerism rate is ≥ 5%.