Multiplexed Quantification of Four Neuroblastoma DNA Targets in a Single Droplet Digital PCR Reaction
Multiplexed Quantification of Four Neuroblastoma DNA Targets in a Single Droplet Digital PCR Reaction
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DOI：
10.1016/j.jmoldx.2020.07.006
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发表时间：
2020-11-01
影响因子：
4.1
通讯作者：
Lodrini, Marco
中科院分区：
文献类型：
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作者：
Peitz, Constantin;Spruessel, Annika;Lodrini, Marco
神经母细胞瘤患者外周血中游离DNA（cfDNA）的检测和表征可作为液体活检的微创方法。分析从血液样品纯化的cfDNA的主要挑战是小样品体积和低cfDNA浓度。液滴数字PCR（ddPCR）是适合于分析低水平cfDNA的技术。本文报告了两种四重ddPCR测定方案，其在单个反应中可靠地定量MYCN和ALK拷贝数以及两种参考基因NAGK和AFF3，并使用cfDNA作为输入准确估计ALK（F1174L）（外显子23位置3522，C>A）和ALK（R1275Q）（外显子25位置3824，G>A）突变等位基因分数。通过调整每个靶分子的探针和引物浓度，优化阳性和阴性液滴的分离，以检测每个ddPCR荧光通道中的两个靶。使用一组10个神经母细胞瘤细胞系和来自9名患者的配对血浆和原发性神经母细胞瘤样品验证四重测定。四重测定中的准确度和灵敏度阈值与相应双链测定中的准确度和灵敏度阈值一致。提供了两种适用于常规临床环境的稳健四重ddPCR方案，仅需要最小血浆体积即可评估MYCN和ALK癌基因状态。
The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALK(F1174L) (exon 23 position 3522, C>A) and ALK(R1275Q) (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.