42P Method optimization for the detection of chimerism by real-time PCR and droplet digital PCR

Abstract

The objective of the present study was to select combinations of primers for genetic polymorphisms (INDELS) used in the analysis of chimerism associated with post-transplant hematopoietic regeneration as well as to test them under real-time PCR (qPCR) and droplet digital PCR (ddPCR) conditions. We searched for genetic polymorphisms, using the EMBL - European Molecular Biology Laboratory - database taking into account certain criteria for selection. There were 85 polymorphisms left at the end of the selection process. For both techniques, we designed primers with the following characteristics: 65°C annealing temperature, amplicon length below 120 bp, at least 3 different nucleotides at the 3’ end between variants and temperature difference between forward and reverse primer of maximum 1°C. Depending on the distribution of genotypes, assays were designed for one or both alleles. Human blood samples from subjects without hematological diseases were obtained in accordance with the Helsinki declaration and genotyped. A novel genotyping protocol, based on 2 reference genes and one allele per polymorphism, was developed. Samples bearing different genotypes were serially diluted to simulate various chimerism levels. In general, for ddPCR increasing primer concentration determines the increase of the amplitude difference between the negative and the positive droplets without affecting the allele specificity. At the same time, the decrease of the annealing extension temperature determined the same phenomenon. Some of the assays showed no differences between 35 and 45 cycles. The new protocol allows the use of smaller sample volumes as compared to traditional genotyping, by reducing reaction number and template quantity. Simulated and obtained chimerism levels showed good correlation between 50% and 1.5% (R2=0.998), the limit of quantification corresponding to 50 molecules. The differences between positive and negative samples are more clearly determined in the case of ddPCR, which confirms that this method is more accurate than the qPCR method. In this context we believe that the ddPCR is a valuable technique for early detection in hematopoietic malignancies. Ioana M. Lambrescu and Victor S. Ionescu - equal contribution.