【摘要】 目的建立检测NK细胞表面免疫球蛋白样受体基因KIR2DS1 mRNA表达水平的荧光定量PCR方法并评价其性能。方法以行异基因造血干细胞移植的供患者共57对为研究对象,针对KIR2DS1基因设计特异性引物及探针,构建TaqMan-MGB荧光定量PCR反应体系,并评价该实验方法的性能,包括:总符合率、重复性、灵敏度及仪器适用范围、技术人员再现性。结果以KIR-SSO基因定性分型法为金标准,通过检测其中35例样品,荧光定量PCR方法对KIR2DS1基因阳性及阴性检测率均为100%。在重复性试验中,选取Ct值分别为高、中、低值3个样品的批内、批间CV分别为0.09%～0.46%、0.71%～1.13%。该方法的灵敏度达10~2 copies/μL,且对拷贝数为10~2 copies/μL的3个样品进行5次重复试验的CV分别为5.37%、2.71%、5.51%。仪器比对结果显示,参比仪器ABI-7500与实验仪器LC-480之间的拟合直线回归方程为Y=0.973 6X+0.118 3(R~2=0.961 9,R~2>0.95)。2名技术人员对10个KIR2DS1基因阳性样品的再现性比对试验结果显示其偏倚(%)均<±5%。结论建立了TaqMan-MGB荧光定量PCR检测KIR2DS1 mRNA表达水平的检测方法,其性能良好。更多还原

【Abstract】 Objective To establish a real-time PCR(RT-PCR) assay for detecting mRNA expression of killer cell immunoglobulin-like receptor(KIR) 2 DS1 gene(KIR2 DS1) on the surface of natural killer(NK) cells, and evaluate its performance. Methods A total of 57 recipient-donor pairs of allogeneic hematopoietic stem cell transplantation(Allo-HSCT) were enrolled in this study. The specific primers and probe of KIR2 DS1 gene were designed for Taqman-MGB fluorescence quantitative PCR detection system. The performance parameters of the detecting system, such as coincidence rate, repeatability, sensitivity, scope of application of the instrument and reproducibility of operation technicians were evaluated and validated. Results The KIR-SSO Genotyping Test was used as the gold standard. The results of 35 samples showed the accuracies of self-built method were all 100% for both of positive and negative KIR2 DS1. Three samples with high, median and low value of Ct values were used to verify the repeatability. The coefficients of variation of intra-assay and inter-assay were ranged from 0.09% to 0.46% and 0.71% to 1.13% respectively. The sensitivity of the established method was up to 10~2 copies/μL at least. The coefficients of variation of the three samples with sensitivity of 10~2 copies/μL were 5.37%, 2.71% and 5.51% in five repeated tests respectively. The regression analysis for the samples measured by ABI-7500 and LC-480 fluorescence quantitative PCR instrument showed regression equation was Y=0.973 6X+0.118 3(R~2=0.961 9, R~2>0.95). The reproducibility of 10 samples with positive KIR2 DS1 operated by two technicians showed that the biases were all less than ±5%. Conclusion A TaqMan-MGB real-time PCR assay for detection of mRNA expression of KIR2 DS1 gene was established successfully with fine performance.更多还原