Establishment of a RT-PCR assay for detection of mRNA expression of KIR2DS1 gene on NK cell surface / 临床检验杂志

ABSTRACT

Objective@#To establish a real-time PCR (RT-PCR) assay for detecting mRNA expression of killer cell immunoglobulin-like receptor (KIR) 2DS1 gene( KIR2DS1 ) 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 KIR2DS1 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 KIR2DS1 . 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 KIR2DS1 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 KIR2DS1 gene was established successfully with fine performance.