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

Abstract

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 ⌬⌬Ct 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 SNPqPCR 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.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR

“…Internal of primers: as an internal control in relative quantitation by AS-qPCR, we used locus-individualized endogenous control with genomic position overlapping with the sequence of the respective informative SNP according to the report by Gineikiene et al [12]. The locus of rs1386718 was used for only the discrimination of donor/recipient pairs, so internal primer has not shown.…”

“…We selected SNPs loci with high minor allele frequency (MAF), which are located on different regions of chromosomes according to the previous studies [12,17,18] and our experiment. In the preliminary study using unrelated 8 pairs, 5 SNPs provided 100% of probability in identifying informative loci in the all 8 pairs.…”

“…The reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 50 cycles of 95°C for 15 s and 60°C for 1 min. As an internal control in relative quantitation by AS-qPCR, we used locus-individualized endogenous controls with a genomic position overlapping the sequence of the respective informative SNPs, according to Gineikiene et al [12]. Relative levels of recipient DNA in the 6 patients post-HSCT were calculated based on the 2 −ΔΔCt method [13].…”

“…Recently, analyses based on real-time PCR using single nucleotide polymorphisms (SNPs) have been reported as alternative methods with more accurate quantification [10][11][12][13]. For detection of an SNP or a single nucleotide mutation, allele-specific PCR (AS-PCR) methods based on an amplification refractory mutation system or a mismatch amplification mutation assay have been developed [14][15][16].…”

“…For detection of an SNP or a single nucleotide mutation, allele-specific PCR (AS-PCR) methods based on an amplification refractory mutation system or a mismatch amplification mutation assay have been developed [14][15][16]. AS-quantitative PCR (AS-qPCR) using a fluorescence probe such as TaqMan probe can quantify the target allele, indicating that AS-qPCR for SNPs has potential to be applied to a chimerism assay following HSCT [10,12].…”