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首页> 外文期刊>Applied Microbiology >High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol
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High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

机译：通过定量末端限制片段长度多态性协议进行高灵敏度稳定同位素探测

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Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)–TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates ( Escherichia coli , Rhodococcus , Variovorax , and Microbacterium ), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.
机译：稳定同位素探测（SIP）已被证明是一种有价值的与培养无关的工具，可将特定的微生物种群与各种自然和工程系统中的选定功能联系起来。但是，将SIP应用于浮力密度相对较小的微生物种群（例如利用化合物作为氮源的种群）会降低标记种群的分辨率。因此，我们开发了串联定量PCR（qPCR）–TRFLP（末端限制性片段长度多态性）方案，该方案通过定量梯度级分中的特定分类组来提高检测分辨率。该方法将控制良好的扩增与TRFLP分析相结合，以定量FAM标记的PCR产物的扩增子库中的相对分类单元丰度，并使用嵌入染料EvaGreen监控扩增。使用以下方法来评估方法的准确性：克隆的16S rRNA基因，从低和高G + C细菌分离株（大肠杆菌，红球菌，Variovorax和微细菌）提取的DNA，以及用已知量的来自细菌分离株。相对于单独的TRFLP分析，使用良好控制的SIP分析已确认，浮力密度微小变化的分辨率得到改善。

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