Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.
Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.
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DOI：
10.3389/fmicb.2017.02017
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发表时间：
2017
影响因子：
5.2
通讯作者：
Or D
中科院分区：
文献类型：
--
作者：
Kleyer H;Tecon R;Or D
基因组测序技术的快速进步使得能够确定相对细菌丰度和群落组成，然而，尽管对于某些生态推论很重要，但物种水平的变化仍然难以检测。我们提出了一种使用基于微流体的定量实时 PCR (qPCR) 提取和直接量化预定义多物种细菌群落的物种组成的方法。我们采用基于通用 16S rRNA 基因预扩增的巢式 PCR 方法，然后使用并行单重 qPCR 反应的微流体阵列检测和定量细菌物种的绝对丰度。目前的微流控 qPCR 支持在单个芯片上同时进行 2,304 个反应，而样品和反应物的自动分配可最大限度地减少移液错误和技术变化。该方法的实用性通过在两种截然不同的环境（沙子微观世界和分批培养）中生长的合成土壤细菌群落来说明。该方案包括总核酸的提取、基因组 DNA 的制备以及细菌群落组成的 qPCR 评估步骤。该方法仅需 2 ng 群落 DNA 即可对细菌物种进行特异性且灵敏的定量。优化的提取方案和预扩增步骤可以快速、定量、同时检测高通量的候选物种。所提出的方法为现有测序方法提供了一种简单而准确的替代方法，特别是当需要物种丰度的绝对值时。物种水平变化的量化有助于从机制上理解特定物种在细菌群落功能中的作用。
Rapid advances in genome sequencing technologies enable determination of relative bacterial abundances and community composition, yet, changes at the species level remain difficult to detect despite importance for certain ecological inferences. We present a method for extraction and direct quantification of species composition of a predefined multispecies bacterial community using microfluidic-based quantitative real-time PCR (qPCR). We employ a nested PCR approach based on universal 16S rRNA gene pre-amplification followed by detection and quantification of absolute abundance of bacterial species using microfluidic array of parallel singleplex qPCR reactions. Present microfluidic qPCR supports 2,304 simultaneous reactions on a single chip, while automatic distribution of samples and reactants minimizes pipetting errors and technical variations. The utility of the method is illustrated using a synthetic soil bacterial community grown in two contrasting environments – sand microcosms and batch cultures. The protocol entails extraction of total nucleic acid, preparation of genomic DNA, and steps for qPCR assessment of bacterial community composition. This method provides specific and sensitive quantification of bacterial species requiring only 2 ng of community DNA. Optimized extraction protocol and preamplification step allow for rapid, quantitative, and simultaneous detection of candidate species with high throughput. The proposed method offers a simple and accurate alternative to present sequencing methods especially when absolute values of species abundance are required. Quantification of changes at the species level contributes to the mechanistic understanding of the roles of particular species in a bacterial community functioning.
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影响因子：
11
作者：
Angel, Roey;Soares, M. Ines M.;Gillor, Osnat
通讯作者：
Gillor, Osnat
影响因子：
4.2
作者：
Angel, Roey;Pasternak, Zohar;Gillor, Osnat
通讯作者：
Gillor, Osnat
影响因子：
11
作者：
Berry, David;Schwab, Clarissa;Loy, Alexander
通讯作者：
Loy, Alexander
影响因子：
16.6
作者：
Jousset, Alexandre;Eisenhauer, Nico;Scheu, Stefan
通讯作者：
Scheu, Stefan
DOI：
10.1073/pnas.82.20.6955
发表时间：
1985-01-01
影响因子：
11.1
作者：
LANE, DJ;PACE, B;PACE, NR
通讯作者：
PACE, NR