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Title: Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation.
Austin Authors: Goh, Su Kah ;Muralidharan, Vijayaragavan ;Christophi, Christopher ;Do, Hongdo;Dobrovic, Alexander 
Affiliation: Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, Victoria, Australia
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia
Issue Date: Mar-2017 2017-01-18
Publication information: Clinical chemistry 2017; 63(3): 742-750
Abstract: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer to Hybridize across an Allelic BREakpoint-PCR). The strategic placement of one primer to hybridize across an allelic breakpoint ensured highly specific PCR amplification, which then enabled the absolute quantification of donor-specific alleles by probe-free ddPCR. dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma. Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection. PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.
DOI: 10.1373/clinchem.2016.264838
ORCID: 0000-0003-3414-112X
PubMed URL: 28100495
Type: Journal Article
Appears in Collections:Journal articles

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