Application of multiple STR-PCR in the detection of chimerism rate after allogeneic hema-topoietic stem cell transplantation
Objective To evaluate the accuracy of quantitative determination of chimerism using GoldeneyeTM20A testing kit,and the impact of stutter peaks on the results.Methods We mixed pairwise blood samples collected from 18 healthy donors to prepare chimera models with different ratios of donor-recipient WBCs.DNA was extracted from the mixed samples,and 20 fluorescence labeled STR loci were amplified using GoldeneyeTM 20A testing kit,and then sequenced by capillary electrophoresis.Finally,the chimerism was calcu-lated in the case of considering(method 2)and ignoring stutter peaks(method 1).Results A total of 63 mixed samples were able to detect alleles of donor recipient specific STR loci,with a minimum detectable cell chimerism rate as low as 0.625%.Only the group with a simulated chimerism rate of 40%showed no statistically significant difference in the results obtained by the two algorithms(P>0.05).When the simulated chimerism rate was≤20%,there was a statistically significant difference in the results obtained by the two algorithms(P<0.05).When using Method 1 for calculation,there was no statistically significant difference between the measured values and theo-retical values of the 40%,20%,and 10%simulated chimeric rate groups(P>0.05).The groups with simulated chi-merism rates of 5%,2.5%,1.25%,and 0.625%showed statistically significant differences between the measured values and theoretical values(P<0.05).When using Method 2 for calculation,there was no statistically significant difference between the measured values and theoretical values of the four simulated chimeric rate groups of 40%,20%,10%,and 5%(P>0.05).The groups with simulated chimerism rates of 2.5%,1.25%,and 0.625%showed statistically significant differences between the measured values and theoretical values(P<0.05).The measured values of the embedding rate obtained by both algorithms show a significant linear correlation with the theoretical values(P<0.05),but the intercept of the regression line in method two on the Y-axis is 0.067,which is closer to 0 than method one at 1.9852.Conclusion Multiple STR-PCR combined with capillary electrophoresis is a sensi-tive assay for detecting chimerism.By selecting ideal loci,and incorporating the stutter peak with the true allele peak,we can obtain more accurate results than by directly calculating and ignoring stutter peaks.
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