共查询到20条相似文献,搜索用时 22 毫秒
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Md. Rashedul Islam Tahera Sultana M. Melvin Joe Jang-Cheon Cho Tongmin Sa 《Saudi Journal of Biological Sciences》2012,19(3):337-342
Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A260/A230 and A260/A280 ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals. 相似文献
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目的:探讨适用于微生物多样性研究的棉田土壤微生物总DNA提取方法。方法:采用4种方法提取不同连作和轮作处理的棉田土壤微生物总DNA,比较其纯度、产率、片段大小,并应用ARDRA技术验证其质量。结果:其中3种方法均可获得23kb的DNA片段,但不同方法提取的DNA的产率和纯度上有明显差异。改良CTAB-SDS法提取的DNA完整性好,得率为24.20μg.g-1干土,纯化后A260/A280和A260/A230为分别为1.80和1.70,纯化回收率可达70.1%,完全适用于后续的PCR分析。结论:采用该法提取棉田土壤总DNA简便而高效。对该法提取获得的棉田土壤微生物总DNA进行ARDRA和DGGE分析,所得图谱能较全面地反映不同处理间微生物多样性及群落结构的差别,为不同栽培体系下棉田土壤微生物的分子生态学研究提供了基础。 相似文献
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Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples" 总被引:1,自引:0,他引:1
Petric I Philippot L Abbate C Bispo A Chesnot T Hallin S Laval K Lebeau T Lemanceau P Leyval C Lindström K Pandard P Romero E Sarr A Schloter M Simonet P Smalla K Wilke BM Martin-Laurent F 《Journal of microbiological methods》2011,84(3):454-460
Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring. 相似文献
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内参基因加标法定量土壤微生物目标基因绝对拷贝数 总被引:1,自引:0,他引:1
【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。 相似文献
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采用4种提取方法对油田上方6个土壤样品微生物总基因组DNA进行提取,并比较了其纯度和浓度。利用定量PCR技术定量分析各土壤中甲烷单加氧酶基因(pmoA)和丁烷单加氧酶基因(brnoX)。与DNA试剂盒法相比,玻璃珠击打法、液氮研磨法、反复冻融法得到DNA纯度较低,需纯化才能进行后续的PCR扩增。液氮研磨法得到的DNA完整性、纯度和得率较其他提取方法均较好,尤其对于生物量较少的深层油田土壤DNA提取较为实用,成本较低。甲烷单加氧酶基因(pmoA)和丁烷单加氧酶基因(bmoX)的定量PCR结果表明,液氮研磨法提取DNA样品的定量结果在气区和油区均出现一定的高值。液氮研磨法较其他方法更适合于油田土壤DNA的提取。下一步研究可以把丁烷氧化茵作为油气指示茵研究中的重点检测对象。 相似文献
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为筛选和建立风沙土中总DNA的提取和纯化方法,选取了5种直接提取法、1种间接提取法和2种纯化法分别对风沙土中总DNA进行了提取和纯化,并对其质量和产量进行了比较.结果表明:6种方法均可从风沙土中提取到大小为23 kb左右的总DNA,其中改进后的高盐提取法(用40%聚乙二醇8000和4 mol·L-1 NaCl沉淀DNA)效果最好,纯化后总DNA的纯度最高,可进行16S rDNA的PCR扩增,且产量仅稍低于试剂盒提取法;电泳加柱回收纯化法的纯化效果较好,经该方法纯化后的总DNA大部分可进行PCR扩增,可满足后续分子操作对DNA纯度的要求. 相似文献
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Determination of whole-genome DNA–DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA–DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA–DNA relatedness between closely related species. 相似文献
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Jing Ning Jost Liebich Matthias Kästner Jizhong Zhou Andreas Schäffer Peter Burauel 《Applied microbiology and biotechnology》2009,82(5):983-993
Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits,
we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as
the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient
gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities
of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected
by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due
to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until
now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced
by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based
methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the
subsequent assessment of microbial community. 相似文献
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Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity 总被引:1,自引:0,他引:1 下载免费PDF全文
The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes. 相似文献
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3 次连续重复提取DNA 能较好反映土壤微生物丰度 总被引:7,自引:1,他引:6
【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。 相似文献
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Effects of agronomical measures on the microbial diversity of soils as related to the suppression of soil-borne plant pathogens 总被引:14,自引:0,他引:14
The diversity of soil microbial communities can be key to the capacity of soils tosuppress soil-borne plant diseases. As agricultural practice, as well as directedagronomical measures, are known to be able to affect soil microbial diversity, it isplausible that the soil microflora can be geared towards a greater suppressivity ofsoil-borne diseases as a result of the selection of suitable soil management regimes.In the context of a programme aimed at investigating the microbial diversity of soilsunder different agricultural regimes, including permanent grassland versus arableland under agricultural rotation, we assessed how soil microbial diversity is affectedin relation to the suppression of the soil-borne potato pathogen Rhizoctoniasolani AG3. The diversity in the microbial communities over about a growingseason was described by using cultivation-based – plating on different media – and cultivation-independent – soil DNA-based PCR followed by denaturing gradient gel electrophoresis (DGGE) community fingerprinting – methods. The results showed great diversity in the soil microbiota at both the culturable and cultivation-independent detection levels. Using cultivation methods, various differences between treatments with respect to sizes of bacterial and fungal populations were detected, with highest population sizes generally found in rhizospheres. In addition, the evenness of eco-physiologically differing bacterial types was higher in grassland than in arable land under rotation. At the cultivation-independent level, clear differences in the diversities of several microbial groups between permanent grassland and arable land under rotation were apparent. Bio-assays that assessed the growth of R. solani AG3 hyphae through soil indicated a greater growth suppression in grassland than in arable land soils. Similarly, an experiment performed in the glasshouse showed clear differences in both microbial diversities and suppressiveness of R. solani growth in soil, depending on the presence of either maizeor oats as the crop. The significance of these findings for designing soil managementstrategies is discussed. 相似文献
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Wen Guo Lan Jiang Shalender Bhasin Shaharyar M. Khan Russell H. Swerdlow 《Mitochondrion》2009,9(4):261-265
Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR. 相似文献
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Quantitative Isolation of Microbial DNA from Different Types of Soils of Natural and Agricultural Ecosystems 总被引:3,自引:1,他引:2
A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R
2= 0.799). The ratio of soil CO2 emission rate to the amount of extractable DNA in the soil was shown to reflect the physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO2. 相似文献
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Effect of soil moisture at different temperatures on Rhizoctonia root rot of wheat seedlings 总被引:1,自引:0,他引:1
The effect of soil moisture at different temperatures on root rot of wheat seedlings caused by Rhizoctonia solani AG-8 was studied in temperature controlled water tanks under glasshouse conditions. Four moisture levels (15, 30, 50 and 75% of soil water holding capacity at saturation which were equal to –10, –7, –5 and –3 kPa, respectively) were tested in tanks maintained 10, 15, 20 or 25 °C. The role of microbial activity in the effect of soil moisture and temperature on disease severity was also studied by including treatments of steam treated soil. Results showed that at soil moisture levels optimum for plant growth (50 and 75% WHC) disease was more severe at a lower temperature (10 °C), but under relatively dry conditions (15% WHC) disease levels were similar at all temperatures tested. In warm soils (20 and 25 °C) at high soil moisture levels (50 and 75% WHC), disease was more severe in steam treated soil than in non-steam treated soil, indicating that the suppression of disease in natural soil under these conditions was associated with high soil microbial activity. 相似文献
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The variation of soil microbial respiration with depth in relation to soil carbon composition 总被引:1,自引:0,他引:1
The vertical variation in soil microbial respiratory activity and its relationship to organic carbon pools is critical for modeling soil C stock and predicting impacts of climate change, but is not well understood. Mineral soil samples, taken from four Scottish soils at different depths (0–8, 8–16, 16–24, 24–32 cm), were analyzed and incubated in the laboratory under constant temperature and environmental conditions. The vegetation type/plant species showed significant effects on the absolute concentration of C components and microbial activity, but the relative distribution of C and respiration rate with soil depth are similar across sites. Soil C pools and microbial respiratory activity declined rapidly with soil depth, with about 30% of total organic carbon (TOC) and dissolved organic carbon (DOC), and about half microbial carbon (Cmic) and respired CO2 observed in the top 8 cm. The ratio of CO2:TOC generally decreased with soil depth, but CO2:DOC was significantly higher in the top 8 cm of soil than in the subsoil (8–32 cm). No general pattern between qCO2 (CO2:Cmic) and soil depth was found. The vertical distributions of soil C pools and microbial respiratory activity were best fitted with a single exponential equation. Compared with TOC and DOC, Cmic appears to be an adequate predictor for the variation in microbial respiration rate with soil depth, with 95% of variation in normalized respiration rate accounted for by a linear relationship. 相似文献
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Camilla E. Thorn Christian Bergesch Aoife Joyce Gustavo Sambrano Kevin McDonnell Fiona Brennan Robert Heyer Dirk Benndorf Florence Abram 《Molecular ecology resources》2019,19(2):439-455
The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co‐recovered from the same biological samples. Commercial kits are currently available for the co‐extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol–chloroform‐based methods for nucleic acids co‐extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost‐effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high‐throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co‐extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram‐positive and Gram‐negative pure cultures. 相似文献
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土壤微生物总DNA提取方法的优化 总被引:1,自引:0,他引:1
【目的】土壤中未培养微生物约占总量的99%,这就意味着绝大多数微生物资源还未得到开发和利用。本研究通过优化土壤微生物总DNA的提取方法,获得较高质量的DNA,为后期研究土壤微生物的多样性及构建大插入片段的宏基因组文库奠定基础。【方法】通过综合比较已报道的微生物DNA提取方法的优缺点,我们设计出一种新的提取方案。对提取过程中的几个关键步骤进行了优化,包括联合使用SDS-CTAB和溶菌酶一起来破细胞,利用氯仿除蛋白,使用PVPP柱纯化DNA等。比较分析了优化后的方法和3种已报道方法所获得的土壤总DNA的产量、纯度及片段大小。【结果】优化后的方法所获得的土壤DNA质量明显有所提高:每克土壤最高能提取95μg DNA,A260/A280和A260/A230比值更接近理想水平,PCR扩增能够得到明显的目标条带,DNA片段最大能达到100 kb左右。【结论】通过比较分析,最终确立了一种较理想的土壤微生物总DNA提取方法,为更好地开发利用土壤未培养微生物资源提供了有力工具。 相似文献