Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury
Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury
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DOI：
10.1373/clinchem.2013.210328
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发表时间：
2013-12-01
影响因子：
9.3
通讯作者：
Schuetz, Ekkehard
中科院分区：
文献类型：
--
作者：
Beck, Julia;Bierau, Sarah;Schuetz, Ekkehard
背景：来自移植受体循环中移植物的无细胞DNA（cfDNA）是排斥反应的潜在生物标志物。通过使用微阵列和供体和受体DNA的大规模平行测序，在心脏移植后的维持阶段研究了其有用性。这些方法的缺点是成本高、周转时间长以及需要供体DNA。因此，我们试图开发一种快速和具有成本效益的方法，使用数字液滴PCR（ddPCR）。方法：血浆样本收集稳定的受体后，肝（LTx，n = 10），肾（KTx，n = 9），心脏（HTx，n = 8）移植，以及从7个额外的患者直接LTx后。已知的单核苷酸多态性被选择为高次要等位基因频率，其中41个水解探针测定被建立。预扩增血浆cfDNA，然后进行常规实时PCR以确定信息性（异源）SNP，然后使用ddPCR将其用于定量（百分比）移植物衍生的cfDNA（GcfDNA）。稳定患者中的GcfDNA是
BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).METHODS: Plasma samples were collected from stable recipients after liver (LTx, n = 10), kidney (KTx, n = 9), and heart (HTx, n = 8) transplantation as well as from 7 additional patients directly after LTx. Known single-nucleotide polymorphisms were selected for high minor allelic frequencies, of which 41 hydrolysis probe assays were established. Plasma cfDNA was pre-amplified, followed by conventional real-time PCR to define informative (heterologous) SNPs, which were then used for quantification (percentage) of graft-derived cfDNA (GcfDNA) using ddPCR.RESULTS: Mean recovery was 94% (SD, 13%) with an imprecision of 4%-14% with the use of controls with 2% minor allele. GcfDNA in stable patients was