Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

Egle Gineikiene^*, Mindaugas Stoskus^* and Laimonas Griskevicius^*

From the Hematology, Oncology, and Transfusion Medicine Center, * Vilnius University Hospital Santariskiu Clinics, Vilnius; and the Clinics of Internal, Family Medicine, and Oncology, Vilnius University, Vilnius, Lithuania

Recently, several studies demonstrated the feasibility of areal-time quantitative PCR (qPCR) approach for chimerism monitoring.qPCR offers a fast, sensitive, and elegant quantification ofgenotypes. However, before it becomes an established methodfor routine chimerism monitoring, a qPCR marker set for everytransplant pair should be available. This requirement posesa major challenge since the genetic markers for qPCR—short insertions/deletions (Indels) and single nucleotide polymorphisms(SNPs)—published to-date do not guarantee applicabilityfor every transplant pair. The aim of our study was to designand validate a new SNP allele-specific system to supplementan already existing Indel primer panel and improve applicabilityof the qPCR approach for chimerism status monitoring. Here,we present an approach for an economical in-house design ofSNP allele-specific qPCR primers/probe sets with a locus-individualizedreference system that allows for the accurate quantificationof the respective informative locus using a simple ${Delta}$ ${Delta}$ Ct method.We designed primers/probe sets specific for seven biallelicSNP loci and validated them in a population of 30 transplantpairs. Repeatability varied depending on the amount of quantifiablegenotype. The combination of our SNP-qPCR system and Indel primersincreased recipient genotype identification from 86.6% to 96.6%when tested in a population of our transplant pairs. These resultsdemonstrate the feasibility of our SNP-based qPCR approach toimprove the applicability of a qPCR for chimerism monitoring.