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quantification of tuber aestivum vittad. mycelium: advantages and limitations of a qPCR approach.

Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach.

机构信息

Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, Czech Republic.

出版信息

Mycorrhiza. 2013 Jul;23(5):341-8. doi: 10.1007/s00572-012-0475-6. Epub 2012 Dec 28.

Abstract

A quantitative real-time PCR (qPCR) marker Ta0 with hydrolysis probe ("TaqMan"), targeted to the internal transcribed spacer region of the ribosomal DNA, has been developed for quantification of summer truffle (Tuber aestivum) mycelium. Gene copy concentrations determined by the qPCR were calibrated against pure culture mycelium of T. aestivum, enabling quantification of the mycelium in soil and in host roots from the fields. Significant concentrations of the fungus were observed not only in the finest roots with ectomycorrhizae but also in other root types, indicating that the fungus is an important component of the microbial film at the root surface. The concentration of T. aestivum in soil is relatively high compared to other ectomycorrhizal fungi. To evaluate the reliability of the measurement of the soil mycelium density using qPCR, the steady basal extracellular concentration of the stabilized T. aestivum DNA should be known and taken into account. Therefore, we addressed the stability of the qPCR signal in soil subjected to different treatments. After the field soil was sieved, regardless of whether it was dried/rewetted or not, the T. aestivum DNA was quickly decomposed. It took just about 4 days to reach a steady concentration. This represents a conserved pool of T. aestivum DNA and determines detection limit of the qPCR quantification in our case. When the soil was autoclaved and recolonized by saprotrophic microorganisms, this conserved DNA pool was eliminated and the soil became free of T. aestivum DNA.

摘要

已开发出一种针对核糖体 DNA 内部转录间隔区的 Ta0 水解探针(“TaqMan”)实时定量 PCR (qPCR) 标记物,用于量化夏季块菌(Tuber aestivum)菌丝体。通过 qPCR 确定的基因拷贝浓度与纯培养的 T. aestivum 菌丝体相校准,使土壤中和田间宿主根系中的菌丝体能够定量。不仅在具有外生菌根的最细根中观察到真菌的显著浓度,而且在其他根类型中也观察到真菌的显著浓度,表明真菌是根表面微生物膜的重要组成部分。与其他外生菌根真菌相比,土壤中 T. aestivum 的浓度相对较高。为了评估使用 qPCR 测量土壤菌丝体密度的可靠性,应了解并考虑稳定的 T. aestivum DNA 的稳定基础细胞外浓度。因此,我们研究了 qPCR 信号在经受不同处理的土壤中的稳定性。田间土壤经过筛分后,无论是否干燥/再润湿,T. aestivum DNA 都会迅速分解。只需大约 4 天即可达到稳定浓度。这代表了 T. aestivum DNA 的保守池,决定了我们情况下 qPCR 定量检测的检测限。当土壤进行高压灭菌并被腐生微生物重新定殖时,这种保守的 DNA 池就会被消除,土壤中就没有 T. aestivum DNA 了。

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