Multiplex real-time quantitative polymerase chain reaction assay for rapid and sensitive detection of hematopoietic...

The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real-time quantitative polymerase chain reaction (RQ-PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ-PCR for MC assessment (six biallelic genetic systems and Y-specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%-0.05%). The efficiencies of multiplex RQ-PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ-PCR vs singleplex RQ-PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ-PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for accurate MC assessment.