Optimization Test Conditions And Evaluation Research Of The Chronic Myeloid Leukemia SsDNA Aptamer Detection Method

Objective:To optimized the testing conditions of the detecting method which set up earlily based on the K562cells ssDNA aptamers detection method and evaluated this method for its clinical chronic myeloid leukemia diagnosis application value.Methods:Used the DNA aptamers by synthesising as templates and designed primers, according to the primers Tm, setting up different annealing temperature gradients, cycle times and the concentration of primers, and so on, optimized respectively the reaction conditions of symmetric and asymmetric PCR,then used the method which set up earlily to make ssDNA aptamers combining with the K562cells, screening out the best combination of ssDNA aptamers and optimizing the testing conditons,finally made use of the best ssDNA aptamers combination combining with the samples of clinical chronic myeloid leukemia and tested,under the optimized testing conditions,according to the relative standards of CLSI, evaluating this method of its methodology,finally reference " the clinical test methodology evaluation", by this method as the candidate method, the gold standard as the reference method,did the methodology evalution research,and use the related indicators as the foundation,drawing the ROC curve to evaluate and research of the method for its clinical diagnosis performance.Results:PCR amplification results dentified by the3%agarose gel electrophoresis,the best annealing temperature of symmetrical PCR was44.2℃, the best cycle was30times, when the proportion of the nonrestrictive primers and restrictive primers concentration was50:1would get the ideal ssDNA and less dsDNA.The best aptamers combination were num.5-Bio-ssDNA aptamer and num.3-FAM-ssDNA aptamers combination. The testing conditions optimization results showed:the optimal formulations of buffer system was8.0gNaCl,0.2gKCl,3.628gNa2HPO412H2O,0.24gKH2PO4,the optimal buffer pH was9.0,the best incubation temperature was37℃and the incubation time was15min,the best FAM-ssDNA aptamer concentration was100pmol,the Bio-ssDNA aptamer concentration was150pmol,cells num-concentration was800～1000×103/mL. To evaluate this method, it had a high sensitivity, low detection threshold value,and a higher specificity, had Linear relationship with predicted values and fitting high, regression equation was Y=1476.4+495.31X, r=0.9766, the intra-assay precision was less than5%, but the days between precision was more than5%, reagent stability analysis results followed:under the condition of-20℃, reagent can be stable placed for3weeks, under the condition of4～8℃can be placed for2weeks, at room temperature only can be for1day. To assess the clinical diagnosis of performance evaluation for this method, the results showed that the best specimen source was venous blood and form was neutrophilsn of extraction, the results of the specimens stability test according to:under the condition of-20℃, specimens can be stable placed for4weeks, under the condition of4～8℃can be placed for5days, under the room temperature can be placed for2days,the average recovery was78.7%,the average interference rate was43.6%.Then tested the fluorescence intensity of each specimens respectively,and calculated the corresponding average and standard deviation,after Independent Samples Test analysis results showed:the case group and the confusable group had no significant difference t=1.227, p=0.287had no statistical significance, the case group and health group had no significant differencet=2.159, p=0.097had no statistical significance, the confusable group and the health group had no significant difference t=1.618,p=0.181had no statistical significance.Finally drew the ROC curve,with sensitivity for the ordinate,with (1-specificity) for the abscissa pictured the curve, calculated the AUC value was0.867,then according to the clinical examination method performance evaluation index and counted the index numerical:the sensitivity was75%,the specificity was77%, the positive likelihood ratio was3.26, the negative likelihood ratio was0.32,the misdiagnosis rate was23%, the missed diagnosis rate was25%, the diagnostic efficiency was0.34,the diagnosis index was152, the reliability was0.306and the availability was0.52.Conclusion:The K562cells ssDNA aptamers detection method which set up earliy had high sensitivity and low detection threshold value,besides had strong specificity, to a certain degree this method provided a new mentality of molecular diagnosis for the chronic myelogenous leukemia;therefore in the clinical diagnosis of performance study for this method, the area under ROC curve was0.867,had higher specificity and sensitivity,had some clinical diagnostic value,but its weak in anti-interference ability, poor in repeatability, its reliability was<0.5,so the clinical application of this method still has some limitations.