Research Group on 'Environmental multi-resistance and efflux pump ', CNRS, Ecole Nationale Vétérinaire de Lyon, and Université Lyon 1, UMR 5557 Ecologie Microbienne, Université de Lyon, 43 boulevard du 11 novembre 1918, 69622, Villeurbanne Cedex, France.
Université de Lyon, France; 'ibio platform', CNRS, Ecole Nationale Vétérinaire de Lyon, and Université Lyon 1, UMR 5557 Ecologie Microbienne, Université de Lyon, 43 boulevard du 11 novembre 1918, 69622, Villeurbanne Cedex, France.
Appl Microbiol Biotechnol. 2016 Dec;100(24):10597-10608. doi: 10.1007/s00253-016-7950-5. Epub 2016 Nov 14.
Droplet digital PCR (ddPCR) allows absolute quantification and tolerance to inhibitors and has been proposed as the method of choice to overcome limitations of qPCR. The aim of this study was to evaluate ddPCR and qPCR performances to detect low copy number and copy number variation of antibiotic resistance genes (sul1 and qnrB genes encoding for resistance to sulfonamides and quinolones, respectively) using bacterial genomic DNA (gDNA) and metagenomic DNA extracted from soil and organic residue samples. With gDNA, qPCR showed a better range of quantification but the lower limit of quantification was at 15 copies of qnrB target vs. 1.6 in ddPCR. In the presence of background DNA or inhibitors, we observed a high loss of sensitivity in qPCR and an overestimation of target sequences. When using high amount of environmental DNA templates (70 ng per reaction), ddPCR was still allowing accurate quantification without adding PCR facilitator (i.e., T4 Gene 32 protein). Sensitivity to detect copy number variation was tenfold higher in ddPCR than in qPCR. Finally, the advantages of using ddPCR in environmental studies were confirmed with the quantification of sul1 and qnrB in soils, manures, or urban wastes.
微滴式数字 PCR（ddPCR）可实现绝对定量，且对抑制剂具有较强的耐受性，已被提议作为克服 qPCR 局限性的首选方法。本研究旨在评估 ddPCR 和 qPCR 在检测抗生素耐药基因（编码磺胺类和喹诺酮类耐药的 sul1 和 qnrB 基因）的低拷贝数和拷贝数变异时的性能，采用细菌基因组 DNA（gDNA）和土壤及有机残留样品中提取的宏基因组 DNA。使用 gDNA 时，qPCR 具有更好的定量范围，但 qPCR 的定量下限为 15 拷贝 qnrB 靶标，而 ddPCR 为 1.6。在存在背景 DNA 或抑制剂的情况下，我们观察到 qPCR 的灵敏度大幅下降，且目标序列被高估。当使用高浓度的环境 DNA 模板（每个反应 70ng）时，ddPCR 仍可在不添加 PCR 促进剂（即 T4 基因 32 蛋白）的情况下进行准确的定量。ddPCR 检测拷贝数变异的灵敏度比 qPCR 高十倍。最后，通过在土壤、肥料或城市废物中定量检测 sul1 和 qnrB，证实了 ddPCR 在环境研究中的优势。
