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1.
Background, aim and scope An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires
extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a
larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still
have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR
inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids
can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg2+ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects
of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were
assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine
plantation of southeast Queensland, Australia.
Materials and methods Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash
pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega
Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to
purify the DNA for PCR amplification, which include PVPP, Sephadex TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification
methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined
by successful PCR amplifications.
Results and discussion The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour
and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions
had been made from neat to 1:103. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration
decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA
losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™
spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and
soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification,
but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in
this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may
be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and
also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification.
Conclusions A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first
introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method
for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification
inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap,
fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely
used to relieve the heavy working load of molecular-biological researchers.
Recommendations and perspectives This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is
effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified
gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect
of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples
which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples. 相似文献
2.
Shin Ugawa Muneyoshi Yamaguchi Satoru Miura Shinji Kaneko 《Soil Science and Plant Nutrition》2013,59(4):510-516
Fine root biomass can be estimated from the quantity of DNA of a target plant extracted from fine root samples using regression analysis. However, the application of this method to fine root samples mixed with soil particles (mixed samples) is difficult due to the high DNA adsorption capacity of some clay minerals. Our aim in this study was to clarify the enhancement level of the DNA extraction efficiency of an improved method, and to obtain a regression line between the amount of DNA and the root biomass from a mixed sample with similar reliability as for fine roots alone (pure root sample). We examined the amount of DNA extracted from a mixture of Zea mays L. fine roots and highly adsorbent Kanuma soil using various concentrations of a skim milk solution, which acts as an adsorption competitor for the soil particles during the DNA extraction process. The amount of DNA of Zea mays extracted from the mixed sample using 0% skim milk was lower than from the pure root sample. However, the amount of DNA extracted from the mixed sample increased with increasing concentrations of skim milk, reaching the same level as for the pure root samples and resulting in a regression line that was similar to the pure root samples. Optimal DNA extraction levels were obtained with the addition of 20?µL of a 20% skim milk solution to 30?mg of a mixed sample. We also discuss the applicability of this method to other plant species and soil types. 相似文献
3.
Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a high-throughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives. 相似文献
4.
In criminal investigations, information on the origin of soils may be crucial for solving cases. The biological complexity of soil may potentially be used for sorting and differentiating between soil samples. Nucleic-acid based analyses of soil microbial populations are powerful tools, routinely used in studies of this habitat. Application of such approaches in forensics implies that a standardized DNA extraction method has to be applied to all samples. In this study, several DNA extraction protocols were compared. An improvement on the method proposed by Tsai and Olson (1991) was found to be most suited to extract DNA from various soil types, including from small samples. A blind test on soils from a crime, an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and denaturating gradient gel electrophoresis for use in forensic science. In most cases, soil patterns clustered according to soil type and location. 相似文献
5.
Although often neglected, variability in cell lysis efficiency and DNA extraction yield represents the major hurdles of any polymerase chain reaction (PCR)-based quantification protocol in soil and other natural environments. In this study we developed a technique that minimizes the effects of these constraints, providing at the same time a reliable internal control to distinguish between PCR-inhibition and negative results. We used Pseudomonas fluorescens Pf153, a root-colonizing bacterium that shows biocontrol activity against tobacco and cucumber black root rot, as the target organism for PCR quantification. Prior to DNA extraction, the genetically engineered, cognate reference strain P. fluorescens CHA0/c2 was inoculated in a reference soil. CHA0/c2 in the reference soil and Pf153 in the soil sample were lysed in parallel and afterward the lysates were mixed in known proportions. CHA0/c2 carries the plasmid pME6031-cmp2 that contains an allelic variant (competitor) of the Pf153 specific sequence Pf153_2. In a quantitative competitive PCR (QC-PCR) assay the competitor allows the quantification of the target strain down to 0.66 Pf153 CFU/mg soil. Processing the reference strain in the same way as Pf153 enables the exact quantification of the target strain in biocontrol assays performed in natural soil, overcoming differences in DNA extraction efficiency and PCR amplification from different soil environments. This technique is easily adaptable to other Pseudomonas strains simply by replacing the competitor used here with one derived from a SCAR-marker which is specific for the strain of choice. 相似文献
6.
Ramos-Gómez S López-Enríquez L Caminero C Hernández M 《Journal of agricultural and food chemistry》2008,56(23):11098-11104
Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis. 相似文献
7.
Extraction of DNA from soil 总被引：1，自引：0，他引：1
Patrick Robe Renaud Nalin Carmela Capellano Timothy M. Vogel Pascal Simonet 《European Journal of Soil Biology》2003,39(4):183-190
There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purification. The alternative approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purification. Several published methods describe the recovery of highly purified nucleic acids that are well-suited for molecular purposes even though a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective. 相似文献
8.
土地混合使用制度下土壤硝态氮分布的地理空间制图研究 总被引：5，自引：0，他引：5
Mapping the spatial distribution of soil nitrate-nitrogen (NO3-N) is important to guide nitrogen application as well as to assess environmental risk of NO3-N leaching into the groundwater. We employed univariate and hybrid geostatistical methods to map the spatial distribution of soil NO3-N across a landscape in northeast Florida. Soil samples were collected from four depth increments (0-30, 30-60, 60-120 and 120-180 cm) from 147 sampling locations identified using a stratified random and nested sampling design based on soil, land use and elevation strata. Soil NO3-N distributions in the top two layers were spatially autocorrelated and mapped using lognormal kriging. Environmental correlation models for NO3-N prediction were derived using linear and non-linear regression methods, and employed to develop NO3-N trend maps. Land use and its related variables derived from satellite imagery were identified as important variables to predict NO3-N using environmental correlation models. While lognormal kriging produced smoothly varying maps, trend maps derived from environmental correlation models generated spatially heterogeneous maps. Trend maps were combined with ordinary kriging predictions of trend model residuals to develop regression kriging prediction maps, which gave the best NO3-N predictions. As land use and remotely sensed data are readily available and have much finer spatial resolution compared to field sampled soils, our findings suggested the effcacy of environmental correlation models based on land use and remotely sensed data for landscape scale mapping of soil NO3-N. The methodologies implemented are transferable for mapping of soil NO3-N in other landscapes. 相似文献
9.
本研究介绍一种分子克隆的新方法。该方法要求在PCR引物设计时,目的片段上游引物的5＇端和下游引物的5＇端各设计约25 bp与目标载体末端同源的序列,或目的载体反向游引物的5＇端和正向引物的5＇端各设计约25 bp与目标片段同源的序列,以便进行融合PCR,PCR反应扩增目的片段后,与载体进行融合PCR。用DpnⅠ消化原始甲基化模板,然后进行转化和重组子鉴定。结果表明利用该方法成功将目的片段插入载体。证明这是一种简便、通用、高效并值得推广的分子克隆的新方法。该方法不需要骨架载体上的特异性酶切位点,特别适用于插入片段中无限制性酶切位点的载体改造;该方法还可以引入定点突变,可便捷构建分析启动子功能等需引入定点突变的载体;该分子克隆方法还可以实现基因的无缝克隆。 相似文献
10.
微生物在众多的自然和人工生态系统中发挥着核心的作用,但能够被培养分离的微生物在大部分生态系统中只占极少一部分,极大地限制了人们对微生物组成、功能及其潜在应用的认识。分子生物学方法,尤其是高通量测序技术应用到微生物生态学研究中,为认识微生物多样性、群落结构组成及其生态功能提供了有利手段。高通量测序作为一种新兴的免培养分子生物学技术,具备检测快速、准确、信息全面丰富等特点。随着高通量测序技术的不断升级换代,测序通量、读长和准确度的不断提升以及成本的大幅下降,该技术在过去十几年间被迅速应用于土壤、水体和肠道等微生物区系的研究中。本文简述了基于高通量测序技术的PCR产物测序技术和宏基因组学测序技术的原理、发展历程、数据分析方法与应用,以及宏基因组学测序技术在病毒学领域的应用,以期为微生物分子生态学研究提供参考。 相似文献
11.
The equivalence of the Calcium‐Acetate‐Lactate and Double‐Lactate extraction methods to assess soil phosphorus fertility
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下载免费PDF全文 Michael van Laak Uwe Klingenberg Edgar Peiter Thomas Reitz Dana Zimmer Uwe Buczko 《植物养料与土壤学杂志》2018,181(5):795-801
A large variety of extraction methods are used worldwide for the estimation of “plant‐available P” in soils. In Germany, the standard extractants are Calcium‐Acetate‐Lactate (CAL) and Double‐Lactate (DL). Until now there is no validated transformation procedure available and studies on the comparability of both methods have reported conflicting evidence. The uncertainty about the equivalence of CAL‐P and DL‐P hinders a direct comparison of the P fertility status and P fertilizer recommendations across Germany. Based on 136 datasets for soil samples from an interlaboratory comparison program and three P fertilization field trial sites, for which plant‐available P had been determined by both the CAL and DL method, we assessed the comparability of both extraction methods and derived simple and multiple regression equations to transform DL‐P into CAL‐P values. On average, DL extracted 30% more P than CAL. However, this strongly depended on soil pH and carbonate content. A simple linear regression model explained 70% of the variance. However, if simple linear regression models were fitted to pH‐specific samples (pH range 4.5 to 7.0) the R2 increased to 0.96. Based on an independent validation dataset (n = 48) we demonstrated that such pH‐specific models were more accurate than models that did not consider pH when transforming DL‐P to CAL‐P values. Multiple regression results showed that out of soil pH, Corg, Nt, and C : N ratio, only soil pH improved the model. The transformation equations in this study provide a step towards an improved comparability of P fertility status assessments of soils across Germany. 相似文献
12.
一种可用于PCR扩增的直接提取土壤细菌DNA的方法 总被引：6，自引：0，他引：6
本文以澳大利亚桉树林和松树林的土壤为例 ,采用Napp提取液和SDS直接溶解土壤细菌 ,并配合温浴 -玻璃珠震荡、苯酚 -氯仿萃取和异丙醇提取以及纯化DNA等步骤 ,直接从土壤样品中提取了土壤细菌DNA。所得DNA完全适用于酶解和PCR扩增的要求。该方法高效简单 ,费用低 ,在土壤微生物研究中具有重要的应用价值 相似文献
13.
The present work describes a method for determining ascorbic acid, which combines iodometry with a voltammetric technique to detect the end point of the titration. In addition, the validity of the method applied to natural vegetable or fruit samples was assessed. The results were compared with those obtained by an accurate method such as HPLC using UV detection. Similar values of ascorbic acid for different natural samples were obtained by means of this approach (p > 0.05). The limit of quantification was 0.1 mg. This technique presents the advantage of other electroanalytical methods such as avoiding filtration or ultracentrifugation steps, with the additional benefit of using the platinum electrodes, which are routinely used in the laboratory. These facts allow a rapid and efficient quantification of ascorbic acid with very low cost of reagents and equipment. 相似文献
14.
Lerat S England LS Vincent ML Pauls KP Swanton CJ Klironomos JN Trevors JT 《Journal of agricultural and food chemistry》2005,53(5):1337-1342
A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract. 相似文献
15.
传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一 相似文献
16.
精细土壤属性信息在诸多领域均具有广泛的应用,历来倍受关注。现有土壤属性预测方法具有适用性不强或需要大量人工经验和专家知识等缺点,限制了这些方法在实际应用中的推广。本文提出了一种土壤属性自适应预测方法,可分为4步:①对采样点进行分组处理;②利用回归模型构建各分组内土壤属性与主导环境因子之间的典型关系;③对分组方案进行自动优化;④利用各组对应的土壤–环境因子典型关系对研究区进行优化拟合预测。为了验证方法的有效性,本文在我国东北典型黑土区以土壤有机质含量为例进行了应用研究,结果表明:所提方法可对环境因子做出自动选择,并可通过优化拟合对土壤属性空间分布进行自适应预测,预测精度较高。本方法初步实现了土壤属性预测的自动化,具有较好的适用性。 相似文献
17.
In this study artificial neural network (ANN) models were designed to predict the biomass and grain yield of barley from soil properties; and the performance of ANN models was compared with earlier tested statistical models based on multivariate regression. Barley yield data and surface soil samples (0–30 cm depth) were collected from 1 m2 plots at 112 selected points in the arid region of northern Iran. ANN yield models gave higher coefficient of determination and lower root mean square error compared to the multivariate regression, indicating that ANN is a more powerful tool than multivariate regression. Sensitivity analysis showed that soil electrical conductivity, sodium absorption ratio, pH, total nitrogen, available phosphorus, and organic matter consistently influenced barley biomass and grain yield. A comparison of the two methods to identify the most important factors indicated that while in the ANN analysis, soil organic matter (SOM) was included among the most important factors; SOM was excluded from the most important factors in the multivariate analysis. This significant discrepancy between the two methods was apparently a consequence ofthe non-linear relationships of SOM with other soil properties. Overall, our results indicated that the ANN models could explain 93 and 89% of the total variability in barley biomass and grain yield, respectively. The performance of the ANN models as compared to multivariate regression has better chance for predicting yield, especially when complex non-linear relationships exist among thefactors. We suggest that for further potential improvement in predicting thebarley yield, factors other than the soil properties considered such as soil micronutrient status and soil and crop management practices followed during the growing season, need to be included in the models. 相似文献
18.
Dried soil samples from many sources have been stored in archives world-wide over the years, but there has been little research on their value for studying microbial populations. Samples collected since 1843 from the Broadbalk field experiment on crop nutrition at Rothamsted have been used to document changes in the structure and composition of soils as agricultural practices evolve, also offering an invaluable record of environmental changes from the pre- to post-industrial era in the UK. To date, the microbial communities of these soils have not been studied, in part due to the well-documented drop in bacterial culturability in dried soils. However, modern molecular methods based on PCR amplification of DNA extracted directly from soil do not require bacterial cells to be viable or intact and may allow investigations into the legacy of bacteria that were present at the time of sample collection.
In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from - and β-proteobacteria as well as firmicutes. PCR using primers specific for ammonia oxidizing bacteria showed similar band profiles across the two treatments in recently collected samples, however older samples from the NPK plot showed greater divergence. Primers specific for the genus Pseudomonas were designed and used in real-time quantitative PCR to indicate that archived soil collected in 1868 contained 10-fold less pseudomonad DNA than fresh soil, representing around 105 genomes g−1 soil. Prior to milling, dramatically less pseudomonad DNA was extracted from recently collected air-dried soil from the NPK compared to the FYM plot; otherwise, the two plots followed similar trends. Overall bacterial abundance, diversity and survival during the archiving process differed in the two soils, possibly due to differences in clay and soil organic matter content. Nevertheless, the results demonstrate that air-dried soils can protect microbial DNA for more than 150 years and offer an invaluable resource for future research. 相似文献
19.
Olivier Couillerot Marie-Lara Bouffaud Ezékiel Baudoin Daniel Muller Jesus Caballero-Mellado 《Soil biology & biochemistry》2010,42(12):2298-2305
Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 104 CFU equivalents g−1 lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 104 CFU equivalents g−1. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants. 相似文献
20.
Hernández M Río A Esteve T Prat S Pla M 《Journal of agricultural and food chemistry》2001,49(8):3622-3627
Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA. 相似文献