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

Abstract

Background: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. Methods: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). Results: Droplet-AS-PCR could determine genotypes within 8 min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. Conclusion: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs

“…For STR-PCR, 20 STR markers (D18S1270, D12S391, D20S161, D11S488, D14S608, D10S2325, D8S306, D9S304, D8S1179, D8S639, D19S253, D16S3253, D21S1437, FGA, D5S818, SE33, TH01, VWF, Penta E, D18S51) were amplified using a GeneAmp PCR System 9700 (Thermo Fisher Scientific, Waltham, MA, USA) as described previously 3 , 16 , 17 . In-house SNP-qPCR was performed with the sets of primers and probes specific for 5 SNPs (rs2385512, rs3769393, rs748235, rs1386718, rs12438539) 18 , using a QuantStudio 3, followed by analysis with QuantStudio Design & Analysis Software v1.4.3 (Thermo Fisher Scientific).…”

Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat

“…The polymerase chain reaction (PCR) is a sensitive and specific technique for detecting small molecular changes. PCR amplification of short tandem repeats (STR), which is already used to assess clinical chimerism following HSCT [9], could be useful for VCA studies as well. STR (also called microsatellites) are repetitive sequences of two to seven base pairs in genomic DNA.…”

“…Although many animal transplantation studies employed flow cytometry and immunohistochemistry for quantitative measurements of chimerism, inconsistency remains as shown at the previous section [3][4][5][6]. On the other hand, other methods have been used to measure chimerism, including fluorescent in situ hybridization of sex chromosomes in sex-mismatched transplantations, PCR amplification of variable number of tandem repeats/short tandem repeats (VNTR/STR), single nucleotide polymorphism (allele-specific PCR) [9], and insertion-deletion biallelic polymerism (indels). The detection limit for STR-PCR is generally around 1% and can be as low as 0.024% with indel-PCR [20].…”

Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

“…Our droplet‐PCR is applicable to various genetic tests . In addition, droplet‐PCR methods using allele‐specific primers (droplet‐AS‐PCR) can detect SNPs …”