Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.
Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.
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DOI：
10.1038/srep25186
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发表时间：
2016-04-28
影响因子：
4.6
通讯作者：
Kaufman BA
中科院分区：
文献类型：
--
作者：
Belmonte FR;Martin JL;Frescura K;Damas J;Pereira F;Tarnopolsky MA;Kaufman BA
线粒体DNA（mtDNA）突变是原发性线粒体疾病的常见原因，并且还与广泛的病症集合有关，包括衰老、神经变性和癌症。这些致病性变异中普遍存在的是mtDNA缺失，其显示出在重链和轻链复制起点之间但不包括重链和轻链复制起点的主要弧中序列丢失的强烈偏好。由于单个mtDNA缺失可以聚集在局部，发生多个混合断点，并在正常的mtDNA序列的存在下，检测广谱突变的方法，提高灵敏度和有限的成本有研究和临床应用。在这项研究中，我们评估了半定量和数字PCR为基础的方法，使用双链参考模板或生物样品的mtDNA缺失检测。我们的目的是描述关键的实验检测参数，这些参数将能够分析疾病进展期间mtDNA缺失负荷的低水平或小差异，并具有有限的假阳性检测。我们确定数字PCR方法通过绝对定量、提高精度和降低测定标准误差显著提高了mtDNA缺失检测灵敏度。
Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.