Real-time Fluorescence Quantitative PCR-SNP Detection Transplant Chimera Method To Establish

Objective: To establish a novel posttransplant assay approach for quantitative assessment of mixed hematopoietic chimerism using real-time polymerase chain reaction (PCR)of single nucleotide polymorphisms (SNPs), and analyse the application of real-time SNP-PCR method for patient treated by allogeneic HSCT. Methods: Eight SNPs which located on six different chromosomes were selected for the identification of recipient and donor-derived cells by SNP-allele specific primers and fluorescent dyes - SYBR Green I. The feasibility of this assay and the accuracy of quantitative results were tested by using serial DNA mixtures from unrelated individuals. Quantitative analysis of the peripheral blood sample collected from 3 patients were analyzed by DNA capillary electrophoresis with multiplex fluorescent short tandem repeats(STR) analysis and the SNP real-time PCR assay. Results: Discrimination between donor and recipient was possible. Dilution experiments of the mock chimerism sample revealed that Ct values correlated linearly with the logarithm of recipient/donor DNA fraction (r>0.98), and the limit of detection for a minor DNA percentage under 0.5 %. The quantitative result coincided with the STR outcomes obtained by capillary electrophoresis combined with fluorescence labeling multiply PCR,and correlated well with the corresponding clinical findings. Conclusion: A real-time SNP-PCR assay may ultimately provide more accurate quantification and shortened turnaround time compared to current post-engraftment assays.