Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

被引:206
|
作者
Prevost-Boure, Nicolas Chemidlin [1 ]
Christen, Richard [2 ,3 ]
Dequiedt, Samuel [4 ]
Mougel, Christophe [1 ,4 ]
Lelievre, Melanie [4 ]
Jolivet, Claudy [5 ]
Shahbazkia, Hamid Reza [6 ]
Guillou, Laure [7 ,8 ]
Arrouays, Dominique [5 ]
Ranjard, Lionel [1 ,4 ]
机构
[1] Univ Bourgogne, INRA, UMR Microbiol Sol & Environm, CMSE, Dijon, France
[2] Univ Nice, Nice, France
[3] Ctr Biochim, CNRS, UMR 6543, Lab Biol Virtuelle, Nice, France
[4] Univ Bourgogne, INRA, CMSE, Dijon, France
[5] INRA, US InfoSol 1106, Orleans, France
[6] Univ Algarve, DEEI FCT, Faro, Portugal
[7] Univ Paris 06, Roscoff, France
[8] CNRS, UMR 7144, Stn Biol Roscoff, Roscoff, France
来源
PLOS ONE | 2011年 / 6卷 / 09期
关键词
POLYMERASE-CHAIN-REACTION; RIBOSOMAL-RNA GENES; MICROBIAL COMMUNITY; BACTERIAL COMMUNITIES; DIVERSITY; DNA; SAMPLES; BIODIVERSITY; REVEALS; ECOLOGY;
D O I
10.1371/journal.pone.0024166
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 (R) 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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页数:13
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