Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia.
Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia.
Clin Chem. 2017 Mar;63(3):742-750. doi: 10.1373/clinchem.2016.264838. Epub 2017 Jan 18.
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 (rimer to ybridize across an llelic akpoint-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.
供体特异性无细胞游离 DNA（dscfDNA）作为监测移植物健康和诊断实体器官移植后移植物排斥的非侵入性生物标志物，其应用日益受到关注。然而，目前用于测量 dscfDNA 的方法可能既昂贵又繁琐。本研究开发了一种无探针的数字液滴 PCR（ddPCR）方法，该方法利用小的缺失/插入多态性（DIP）来规避这些限制，同时不影响 dscfDNA 的定量。该方法称为 PHABRE-PCR（rimer to ybridize across an llelic akpoint-PCR）。通过将一个引物设计成横跨等位基因断点进行杂交，确保了高度特异性的 PCR 扩增，然后通过无探针 ddPCR 对供体特异性等位基因进行绝对定量。
对 3 例肝移植受者进行了 dscfDNA 的连续测量。首先对供体和受者基因组 DNA 进行了一组 DIP 基因分型，以鉴定供体特异性等位基因。然后选择区分供体特异性和受体特异性 DNA 的等位基因，以定量受者血浆中的 dscfDNA。
未扩增非靶向等位基因证实 PHABRE-PCR 具有高度特异性。在接受移植的受者中，dscfDNA 在第 3 天增加，但在 2 周内无并发症的 2 例受者中，dscfDNA 减少并在低浓度下稳定。在第 3 例移植受者中，dscfDNA 的明显增加与移植物排斥发作同时发生。
PHABRE-PCR 能够高度特异性和灵敏地定量 dscfDNA。基于 DIP 的方法的实施允许监测 dscfDNA，作为实体器官移植后移植物健康的潜在衡量标准。
