Kabir Shaila, Rajendran Narasimmalu, Amemiya Takashi, Itoh Kiminori
Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
J Biosci Bioeng. 2003;96(4):337-43. doi: 10.1016/S1389-1723(03)90133-2.
Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount ofA. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships (R2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 x 10(9) copies/microl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 x 10(8) and 2.7 x 10(8) copies/microl for woodland and grassland, respectively), high DNA yield (6.4 microg/g and 1.8 microg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.
对环境中的真菌种群进行定量分析，对于更好地理解各种微生物过程至关重要。最近，实时定量PCR（RTQ-PCR）技术的发展消除了与传统定量PCR相关的变异性，从而使PCR产物的常规可靠定量成为可能。因此，在本研究中，RTQ-PCR被用于对通过三种常用DNA提取方案（珠磨匀浆法、液氮研磨法以及基于热去污剂SDS的酶解结合珠磨法）提取的真菌目标DNA进行定量，以确定目标DNA定量的适用性。为了本研究的目的，选择烟曲霉纯培养物（模式生物）、接种了已知量烟曲霉的灭菌土壤（模式土壤系统）以及林地和草地土壤样本（环境样本），通过上述三种不同方案提取DNA。然后通过光谱学和RTQ-PCR系统对提取的DNA进行定量。使用18S rDNA特异性通用真菌引物对目标部分进行定量，然后通过琼脂糖凝胶电泳验证扩增产物。用于RTQ-PCR定量的标准曲线显示出很强的线性关系（引物对NS1和NS2的R2 = 0.9994，引物对nu-SSU-0817和nu-SSU-1196的R2 = 0.9938），且扩增效率较高，引物对NS1和NS2的e = 0.983，引物对nu-SSU-0817和nu-SSU-1196的e = 0.956。尽管对于纯培养物，基于热去污剂SDS的酶解结合珠磨法显示出最高的目标DNA拷贝数（1.5×10⁹拷贝/微升），但对于模式土壤系统和两个环境样本，基于高目标DNA拷贝数（林地和草地分别为6.16×10⁸和2.7×10⁸拷贝/微升）、高DNA产量（林地和草地土壤分别为6.4微克/克和1.8微克/克）以及基于目标DNA拷贝数的高回收率（39.2%），发现珠磨法是合适的，表明总体提取效率较高。
