Single nucleotide polymorphism-based system improves the applicability of quantitative PCR for chimerism monitoring.

Recently, several studies demonstrated the feasibility of a real-time quantitative PCR (qPCR) approach for chimerism monitoring. qPCR offers a fast, sensitive, and elegant quantification of genotypes. However, before it becomes an established method for routine chimerism monitoring, a qPCR marker set for every transplant pair should be available. This requirement poses a major challenge since the genetic markers for qPCR--short insertions/deletions (Indels) and single nucleotide polymorphisms (SNPs)--published to-date do not guarantee applicability for every transplant pair. The aim of our study was to design and validate a new SNP allele-specific system to supplement an already existing Indel primer panel and improve applicability of the qPCR approach for chimerism status monitoring. Here, we present an approach for an economical in-house design of SNP allele-specific qPCR primers/probe sets with a locus-individualized reference system that allows for the accurate quantification of the respective informative locus using a simple DeltaDeltaCt method. We designed primers/probe sets specific for seven biallelic SNP loci and validated them in a population of 30 transplant pairs. Repeatability varied depending on the amount of quantifiable genotype. The combination of our SNP-qPCR system and Indel primers increased recipient genotype identification from 86.6% to 96.6% when tested in a population of our transplant pairs. These results demonstrate the feasibility of our SNP-based qPCR approach to improve the applicability of a qPCR for chimerism monitoring.