Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR.
Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR.
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DOI：
10.1371/journal.pone.0024166
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Ranjard L
中科院分区：
文献类型：
--
作者：
Chemidlin Prévost-Bouré N;Christen R;Dequiedt S;Mougel C;Lelièvre M;Jolivet C;Shahbazkia HR;Guillou L;Arrouays D;Ranjard L
真菌是土壤生物多样性和功能的重要组成部分。然而，真菌群落的表征和知识受到阻碍，因为很少有引物组可用于通过实时定量PCR（实时Q-PCR）来量化真菌丰度。本研究的目的是通过使用SYBRGreen®方法将已经用于研究土壤真菌群落遗传结构的引物组并入实时Q-PCR中来量化土壤中的真菌丰度。为了满足实时定量PCR的要求，提高检测技术的准确性和重复性，本研究对18 S rRNA基因保守区进行了研究。这些区域几乎不受长度多态性的影响，并且可以提供足够小的靶标，这是提高检测技术的准确性和再现性的关键标准。对靶向18 S rRNA基因的33个引物组进行了计算机模拟分析，以选择具有最佳实时Q-PCR潜力的引物组：短扩增子长度;良好的真菌特异性和覆盖率。在检测的33组中，特异性、覆盖度和扩增子长度之间的最佳一致性是引物组FR 1/FF 390。FR 1/FF 390特异性的计算机模拟分析也为先前发表的该引物组分析提供了额外信息。通过对从对5个独立土壤样品进行的真实的Q-PCR测定中获得的扩增子进行克隆-测序，在体外验证引物组FR 1/FF 390对真菌的特异性。该试验还用于评价该方法的灵敏度和重现性。最后，通过对24个不同理化和环境特征的土壤样品中真菌丰度的测定和排序，确定了土壤质地、有机碳含量、C∶N比和土地利用方式在确定土壤真菌丰度中的重要性。
Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.
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