Molecular quantification of environmental DNA using microfluidics and digital PCR
Molecular quantification of environmental DNA using microfluidics and digital PCR
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DOI：
10.1016/j.syapm.2012.06.006
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发表时间：
2012-09-01
影响因子：
3.4
通讯作者：
Inagaki, Fumio
中科院分区：
文献类型：
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作者：
Hoshino, Tatsuhiko;Inagaki, Fumio
实时荧光定量PCR已被广泛应用于自然微生物生境中基因丰度的评估。然而，PCR抑制物质常常降低PCR的效率，导致低估靶基因的拷贝数。使用微流体的数字PCR是一种允许绝对定量DNA分子的新方法。本研究将数字PCR应用于环境样品，并测试了PCR抑制剂对DNA定量的影响。在控制实验中使用X DNA和腐殖酸，低估gimel DNA在1/4400的理论值，观察到6.58纳克亩L-1腐殖酸。相比之下，数字PCR提供了准确的定量数据与浓度的腐殖酸高达9.34纳克亩L-1。还测试了稻田土壤提取物对古细菌165 rRNA基因定量的抑制作用。通过稀释DNA提取物，来自实时PCR和数字PCR的定量拷贝数变得相似，表明稀释是补救PCR抑制的有用方式。然而，稀释策略并不适用于所有自然环境样品。例如，当测试海洋地下沉积物样品时，通过数字PCR，古细菌16 S rRNA基因的拷贝数为1.04 x 103拷贝/g沉积物，而实时PCR仅产生4.64 x 102拷贝/g沉积物，这很可能是由于抑制作用。本研究的数据表明，抑制性物质对微流控和数字PCR的DNA定量几乎没有影响，显示了数字PCR在准确定量从各种微生物栖息地提取的DNA方面的巨大优势。(c)2012 Elsevier GmbH. All rights reserved.
Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using X DNA and humic acids, underestimation of gimel DNA at 1/4400 of the theoretical value was observed with 6.58 ng mu L-1 humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng mu L-1. The inhibitory effect of paddy field soil extract on quantification of the archaeal 165 rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04 x 10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64 x 102 copies/gsediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats. (c) 2012 Elsevier GmbH. All rights reserved.