Detection of low abundance human health pathogens in environmental samples is a challenge for water monitoring. This limitation can be overcome by the introduction of multiple displacement amplification (MDA) where a minute amount of genetic material can be amplified using a phi-29 DNA polymerase. However, the genetic makeup and the concentration of the polynucleotides might influence the amplification process due to inherent assay bias. Herein, a series of experiments were designed to demonstrate the effect of genome length, guanidine and cytosine content, and template concentration on the efficiency of MDA. Quantitative polymerase chain reaction (qPCR) was performed to quantify pre- and post-MDA concentrations of selected genes. Linear regression between pre- and post-MDA log gene copies L⁻¹ of both environmental and lab-grown samples showed a positive correlation (F = 77.59, P < 0.001, R² = 0.7, slope = 1.01). Correlation between relative polynucleotide increase after MDA and target organism length and gene target guanidine and cytosine (G + C) content (F = 4.3, P = 0.02) shows that lower G + C and higher genome length is favored in the MDA process. The MDA process was shown to favor a longer genome over a shorter genome (1.19 and 1.04 change in log gene copy L⁻¹, respectively) and a lower G + C content over a higher G + C content (1.11 and 0.61 change in log gene copy L⁻¹, respectively). There was no MDA bias observed when polynucleotides had the same G + C and genome length but different initial concentrations. This study highlights the need for increased caution when interpreting relative abundance of organisms amplified by MDA such as in next generation sequencing.

在环境样品中检测低丰度人类健康病原体是水监测的一个挑战。这种限制可以通过引入多重置换扩增 (MDA) 来克服，其中可以使用 phi-29 DNA 聚合酶扩增微量的遗传物质。然而，由于固有的分析偏差，基因组成和多核苷酸的浓度可能会影响扩增过程。在此，设计了一系列实验来证明基因组长度、胍和胞嘧啶含量以及模板浓度对 MDA 效率的影响。进行定量聚合酶链反应 (qPCR) 以量化所选基因的 MDA 前后浓度。MDA 前和后对数基因拷贝 L ^-1之间的线性回归环境样本和实验室培养样本的比例均呈正相关（F = 77.59，P  < 0.001，R ²  = 0.7，斜率 = 1.01）。MDA 后相对多核苷酸增加与靶标生物体长度和基因靶标胍和胞嘧啶 (G + C) 含量 (F = 4.3, P  = 0.02) 之间的相关性表明，在 MDA 过程中，较低的 G + C 和较高的基因组长度是有利的。MDA 过程显示出有利于较长基因组而不是较短基因组（log 基因拷贝 L ^{-1 的}变化分别为1.19 和 1.04 ）以及较低 G + C 含量优于较高 G + C 含量（log 基因拷贝 L ^{-1 的}变化为 1.11 和 0.61）基因拷贝 L ^-1， 分别）。当多核苷酸具有相同的 G + C 和基因组长度但不同的初始浓度时，没有观察到 MDA 偏差。这项研究强调了在解释由 MDA 扩增的生物体的相对丰度时需要更加谨慎，例如在下一代测序中。