Circulating cell−free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time−consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non−clinical context. Considering International Organization for Standardization, as well as bioanalytical method validation guidelines we provide a comprehensive procedure to validate assays for cfDNA quantification from unpurified blood plasma. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus. The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI: 8.1−20.3), and intermediate precision ≤ 12.1% (95% CI: 9.2−17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ≈2 fold after all out walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting and can be used as a standard to control pre−analytical factors including cfDNA losses during purification.

中文翻译：

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无需通过DNA提取即可验证定量PCR分析以进行无细胞DNA检测：系统性红斑狼疮患者的运动诱发动力学

循环无细胞DNA（cfDNA）已作为许多疾病的筛选工具进行了研究。为了避免昂贵且费时的DNA分离，可以建立直接定量PCR分析。但是，需要严格的验证才能在临床和非临床环境中提供可靠的数据。考虑到国际标准化组织以及生物分析方法验证指南，我们提供了一种综合程序来验证未纯化血浆中cfDNA定量的检测方法。验证了90 bp和222 bp的检测方法，以研究系统性红斑狼疮患者运动后cfDNA的动力学。分析显示超低定量限（LOQ）为0.47和0.69 ng / ml，重复性≤11.6％（95％CI：8.1-20.3），中间精度≤12.1％（95％CI：9.2-17.7）。进行的样品重新分析证实了该过程的准确性。分析前因素的其他考虑表明，离心速度和温度不会改变cfDNA浓度。在SLE患者中，所有步行锻炼后cfDNA均增加≈2倍，休息60分钟后恢复正常。建立的检测方法可在临床环境中对微量血浆中的cfDNA进行可靠且经济高效的定量分析，并可作为控制纯化前cfDNA丢失等分析前因素的标准。