Mitochondria are vulnerable to the effects of ionizing radiation; damage to mitochondrial DNA (mtDNA) may be more extensive and persistent than damage to nuclear DNA (nDNA). Variation in mtDNA copy number has been proposed as a marker for mitochondrial dysfunction in response to ionizing radiation. We have developed a precise and sensitive duplex droplet digital PCR (ddPCR) method for quantitation of the mtDNA/nDNA ratio in the model organism Caenorhabditis elegans. The effect on this ratio was investigated over a wide range of doses (0.03–72 Gy) of chronic gamma irradiation. Five mitochondrial targets and two nuclear reference genes were amplified pairwise in duplex PCR format (one mitochondrial and one nuclear target per PCR) by both ddPCR and quantitative PCR (qPCR). The results showed that ddPCR but not qPCR enabled detection of a significant increase in mtDNA copy number (1.6 ± 0.1-fold) for nematodes exposed to high doses (≥24 Gy). Thus, ddPCR provided higher precision and greater sensitivity than qPCR for detection of mtDNA copy number variation. The variation followed a Hill-type dose response with threshold 10.3 ± 1 Gy. This strongly suggests that chronic genotoxic stress affects mtDNA replication. The duplex ddPCR method is a novel, high-precision, sensitive tool for determination of mitochondrial DNA copy number variation and function in C. elegans.

线粒体容易受到电离辐射的影响；对线粒体 DNA (mtDNA) 的损害可能比对核 DNA (nDNA) 的损害更为广泛和持久。已提出 mtDNA 拷贝数的变化作为响应电离辐射的线粒体功能障碍的标志。我们开发了一种精确且灵敏的双链液滴数字 PCR (ddPCR) 方法，用于对模型生物秀丽隐杆线虫中的 mtDNA/nDNA 比率进行定量. 在大范围剂量 (0.03–72 Gy) 的慢性 γ 辐射中研究了对该比率的影响。通过 ddPCR 和定量 PCR (qPCR) 以双重 PCR 格式（每个 PCR 一个线粒体和一个核靶标）成对扩增了五个线粒体靶标和两个核参考基因。结果表明，ddPCR 而非 qPCR 能够检测到暴露于高剂量（≥24 Gy）的线虫的 mtDNA 拷贝数显着增加（1.6 ± 0.1 倍）。因此，在检测 mtDNA 拷贝数变异方面，ddPCR 比 qPCR 提供了更高的精度和更高的灵敏度。变化遵循 Hill 型剂量反应，阈值为 10.3 ± 1 Gy。这强烈表明慢性基因毒性应激影响 mtDNA 复制。双链 ddPCR 方法是一种新型、高精度、C. 线虫。