Short Term Influence of Organic and Inorganic Fertilizer on Soil Microbial Biomass and DNA in Summer and Spring

The present study was conducted to see the short term impact of organic and inorganic fertilizers on soil microbial biomass both in spring and summer. Also aimed to observe the correlation between soil microbial biomass and soil DNA. The study concluded that type of fertilizer might alter the soil microbial biomass and DNA contents. In soil treated with organic fertilizers resulted in higher concentrations of microbial biomass and DNA contents in summer as compared to spring dute to increase in temperature. Correspondingly, in case of inorganic fertilizer, concentrations of soil microbial biomass and DNA detected higher in summer instead of spring. The statistical correlation between soil microbial biomass, DNA and ODR in spring and summer along with organic and inorganic fertilizers were calculated highly significant （p〉0.01）. This study demonstrated the impact of fertilizers and seasonal variations on soil microbial biomass and also revealed significant correlation between soil microbial biomass and soil DNA.

DNA Damage Caused By Pesticide-contaminated Soil

Objective To determine the DNA damaging potential and the genotoxicity of individual compounds in pesticide contaminated soil. Methods In the present study, DNA damaging potential of pesticide-contaminated soil and the genotoxicity of individual compounds present in the soil were assessed using fluofimetdc analysis of DNA unwinding assay. Results The contaminated soil sample showed 79% （P〈0.001） of DNA strand break, whereas technical grade of major catbaryl and α-naphthol constituents of the contaminated soil showed 64% （P〈0.01） and 60% （P〈0.02） damage respectively. Conclusion Our results indicate that the toxicity caused by contaminated soil is mainly due to carbatyl and α -napthol, which are the major constituents of the soil sample analyzed by CrC-MS.

Strategies for the Extraction, Purification and Amplification of Metagenomic DNA from Soil Growing Sugarcane

Recently, studies were initiated to investigate the metagenome, which represents the genomes of cultured and uncultured microbes, as a rich source for isolation of many novel genes. The meta-genomic approach originated from the molecular analysis of microbial communities, which revealed that the majority of microorganisms in nature were not cultivable by standard culturing techniques. Therefore, most microorganisms in nature have not been characterized. Although numerous methods have been reported for direct DNA isolation and purification from microorganisms in soil, the sample preparation procedures and experimental conditions used in different studies vary widely. Soils are therefore one of the most challenging environmental matrices from which to obtain microbial DNA that will support PCR. The Papaloapan River is the second largest river basin in México. For the climatic conditions of this region, there is great diversity in plants, animals and microorganisms. In the Papaloapan region different fruits are grown, however, the main crops are sugarcane and pineapple. In this work the extraction of DNA from soils of sugarcane cultivation was performed. We used PCR tests to assess the quality of DNA extracted from soil by amplifying the 16S rDNA gene. Changes in both protocols were performed;satisfactory results were obtained as to the quality of DNA and gene amplification. These results will allow continuing the metagenomic studies, such as sequencing, library construction and identification of enzymes cellulase and amylase activity. It is the first time these studies were performed in the Papaloapan region.

Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content

The aim of the study was the statistical evaluation of the impact of water potential (pF), oxygen availability (ODR) and the way of land use on microbial biomass (MB) and soil DNA content. Soil was extracted from the surface (0 - 20 cm) and subsurface (20 - 40 cm) layers of Mollic Gleysol. Soil material was collected in July 2009 from the village Kosiorow (SE part of Poland), from the two distinct neighbouring areas: agriculturally exploited (AE), and fallow land (FL), which served as the control area. Moisture content was determined for a range of pF values (0, 1.0, 1.5, 2.0), which corresponded to availability of water usable by microorganisms and plant roots. Finally, our results revealed significant (p < 0.001) positive relationship between DNA and soil MB content, and negative correlations between soil MB and both pF and ODR. Importantly, MB seemed significantly dependent on the different way of land use, and higher MB content was noted in the soil agriculturally exploited (p < 0.05) in contrast to the control area.

Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different minerals （goethite, kaolinite, montmorillonite）. DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

DNA extraction method selection for agricultural soil using TOPSIS multiple criteria decision-making model

There is an increased interest in the extraction of nucleic acids from various environmental samples since culture-independent molecular techniques contribute to deepen and broaden the understanding of a greater portion of uncultivable microorganisms. Due to difficulties to select the optimum DNA extraction method in view of downstream molecular analyses, this article presents a straightforward mathematical framework for comparing some of the most commonly used methods. Four commercial DNA extraction kits and two physical-chemical methods (bead-beating and freeze-thaw) were compared for the extraction of DNA under several quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A260/230 ratios), degradation degree of DNA, easiness of PCR amplification, duration of extraction, and cost per extraction. From a practical point of view, it is unlikely that a single DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) was employed to compare the methods. The PowerSoil? DNA Isolation Kit was systematically defined as the best performing method for extracting DNA from soil samples. More specifically, for soil:manure and soil:manure:biochar mixtures, the PowerSoil?DNA Isolation Kit method performed best, while for neat soil samples its alternative version gained the first rank.

Combined Effects of Cadmium and Phenanthrene on DNA Sequence Diversity of Soil Microbial Community

The DNA sequence diversities for microbial communities in soils contaminated by heavy metal (HM) Cadmium (Cd) and polycyclic aromatic hydrocarbon (PAH) phenanthrene were evaluated by Random Amplified Polymorphic DNA (RAPD) analysis in pot and laboratory experiments. Fourteen random primers were used to amplify RAPDs from microbial community DNAs in four soils contaminated by HM or/and PAH. The products of 14 primers were separated in gel and generated 212 reliable fragments, of which 203 were polymorphic. The numbers of microorganisms (fungi, bacteria and actinomycetes) were also analyzed. The results showed that the total number of microorganisms in the soil polluted by Cd was lowest among the soils, about 67.4 % lower than the soil without pollution, especially the number of bacterial, about 73.8 %. In contrast, there was the highest number of microorganisms in the soil polluted by phenanthrene, about 180 % more than those in the control without pollution, especially the number of actinomycete, about 19 times of control. The number of microorganism of soil polluted by Cd + phenanthrene was fallen between Cd and phenanthrene, about 19.5 % lower than control. The number of fungi showed no significant differences among soils. However, increases in DNA sequence diversity were observed in all contaminated soil samples. The effects were stronger in the case of soil contaminated with PAH and HM than those in soils amended with HM or PAH, separately. The soil with Cd and phenanthrene had the highest richness, modified richness and Shannon-Weaver index. These results showed that HM or/and PAH might result in some changes for soil microbe’s DNA itself, such as sequence breakage or insertion and genomic rearrangement. The RAPD marker technique combined with traditional methods appears to be an effective approach for studying the combined effects of HM and PAH on DNA sequence diversity of soil microbial community.

土壤微生物总DNA的提取和纯化

本文建立了从土壤中提取总DNA的方法 ,并通过改进使适合于对革兰氏阳性菌的提取。用 9种性质不同的土壤进行验证 ,均提取到了DNA ,每克干土的DNA提取量从 3 30 μg～1 3 41 μg,通过透析袋回收进行纯化 ,纯化回收率达到 65 34% ,纯化后的土壤DNA可以直接扩增出 1 6SrDNA。 9种土壤的提取率从 60 5 1 %～ 93 45 % ,可以从每g干土添加

从土壤中提取DNA用于PCR扩增

设计、比较了5种直接从土壤中提取DNA的方法。实验结果表明这5种方法都可以从土壤中提取到长度大于15kb的DNA片段,但在不同方法间DNA的产量存在很大差异;初提的土壤DNA经进一步提纯后均可用于PCR反应,利用细菌16S rRNA基因和抗菌肽Shiva-1基因的引物都得到了相应的目的产物。其中方法5提取DNA产量最高,无明显降解,且重复性好,是一种从小量土壤样品中直接提取DNA的理想方法。

土壤微生物分子生态学研究中总DNA的提取

建立了一种土壤DNA提取方法。根据DNA产量和纯度2个评价指标,从3种手提土壤DNA方法中优选出方法B为手提DNA方法(Labmethod),它包括样品预处理,细胞裂解,粗DNA纯化,其中细胞裂解组合了玻璃珠击打,SDS裂解,溶菌酶裂解。进一步应用PCR-限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)技术及PCR-温度梯度凝胶电泳(Temperaturegradientgelelectrophoresis,TGGE)技术,结合DNA产量、纯度、片段大小以及所反映的微生物群落结构特性等指标评价了手提方法(Labmethod)得到的总DNA质量,并将这些结果与2种应用较广的商业试剂盒(MoBioUltraCleanSoilDNAKit和Bio101FastDNASPINKit(ForSoil))所得DNA的各种指标进行了比较。结果表明,手提方法(Labmethod)的粗DNA产量低于Bio101Kit的,但高于MoBioKit的。这些方法所得DNA的长度都在21kb左右,Labmethod提取的DNA没有严重被剪切现象,而2种试剂盒提取的DNA都有不同程度的剪切。手提方法得到的DNA经纯化后,应用细菌及真菌特异引物进行PCR扩增,均能获得目的片段,表明该方法能从土壤中同时有效提取细菌和真菌总基因组DNA。并且,手提方法所得DNA的细菌16SrDNA和真菌18SrDNA的PCR-RFLP图谱与两种商业试剂盒的图谱基本相似,但3种方法提取的DNA在细菌16SrDNAV3区和真菌28SrDNA片段的PCR-TGGE图谱上都存在一定差异,主要表现在一些弱势条带的有无或强弱上。这说明手提方法提取的总DNA与2种商业试剂盒一样,在一定程度上能反映微生物群落的多样性和组成。总之,手提DNA方法(Labmethod)所用试剂普通,价格便宜,能在4h以内获得够质够量的土壤DNA用于微生物分子生态学研究,较适合于广大普通实验室进行土壤DNA提取工作。

浙贝母根际土壤总DNA提取和纯化方法的比较

本研究通过对6种土壤总DNA提取方法(CTAB-SDS-冻融法、玻璃珠-SDS-酚氯仿抽提法、玻璃珠-聚乙烯聚吡咯烷酮-SDS法、玻璃珠-聚乙烯聚吡咯烷酮-溶菌酶法、玻璃珠-聚乙烯聚吡咯烷酮-冻融法和UltraClean试剂盒法)和4种纯化方法(改进琼脂糖凝胶电泳法、2%聚乙烯吡咯烷酮-琼脂糖凝胶电泳法、PVPP层析柱法和低熔点琼脂糖电泳法)的比较,证明利用20 mmol/L EDTA(pH 7.5)预处理土壤后,利用CTAB-SDS-冻融法提取土壤总DNA并经改进琼脂糖凝胶电泳法纯化获得的浙贝母根际土壤总DNA的得率和纯度相对较高,达44.00μg/g±2.65μg/g土壤,可用于后续基于16S rDNA分析基础上的土壤微生物分子生态学的分析工作。

用于分子生态学研究的土壤微生物DNA提取方法

利用SDS高盐法和变性剂加SDS高盐法对土壤微生物总DNA进行了提取,然后通过电泳加树脂柱回收和连续2次树脂柱回收方法进行了纯化.结果表明,变性剂加SDS高盐法的DNA提取效率明显高于前者,电泳加树脂柱法的纯化效果更好.通过PCR扩增表明,经过纯化后的DNA,都可以进行16SrDNA扩增和nirK、nosZ、nifH等功能基因的扩增.因此,变性剂加SDS高盐法是一种更为高效、可靠且适合于环境微生物分子生态学研究的DNA提取方法.

土壤微生物DNA提取方法的研究

目的研究土壤微生物DNA的提取方法。方法选用3种常见有效的土壤微生物DNA提取方法与本实验设计的土壤微生物DNA提取方法对麦田土壤微生物DNA进行提取比较。结果4种方法均能从麦田土壤中提取到片段长度大于22kb的DNA片段,不同方法提取的DNA浓度有一定的差异。提取得到的总DNA不需要纯化就可以用于PCR扩增,使用细菌16S rDNA的通用引物可以扩增得到相应的片段。结论该法操作简便、提取快速、DNA纯度和得率较高。

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。

土壤细菌DNA的抽提及分析

应用不同的方法从土壤中分离细菌DNA ,以紫外分光光度法及琼脂糖凝胶电泳法分析所得细菌DNA的纯度与得率 ,比较不同的细菌收集缓冲液、细菌裂解缓冲液、细菌裂解过程及不同的抽提修饰步骤对土壤细菌DNA抽提结果的影响 ,确定了最佳的土壤细菌DNA抽提方案 .以该方案对土壤细菌DNA进行了大规模的抽提 ,所得产物的A2 60 /A2 80 为 1.6 8,A2 60 /A2 3 0 为 0 .87,得率w(DNA/干土 )为 1.90 μg/g .在土壤细菌中加入 1μg标准λDNA进行了 3次回收实验 ,平均回收率为 82 .33%.

高盐极端环境土壤基因组DNA的分离纯化方法研究及基因文库的构建

以钦州市犀牛角镇晒盐池内（盐浓度〉25％）的土壤样品为研究对象，对土壤混合基因组DNA的提取和纯化方法进行了深入研究，结果表明。冻融法、十二烷基磺酸钠（SDS）和蛋白酶K联合使用的提取法，能有效提高DNA得率，DNA的产量达到每克土壤样品10～15μg。交联聚乙烯吡咯烷酮（PVPP）、SeDhadex G-200树脂以及大量胶回收的使用，可有效纯化DNA。纯化的DNA产量可达每克土壤样品4～6μg，DNA片段大小〉30kb，将纯化的DNA用BamH Ⅰ部分酶切后构建以pLAFR3为载体的混合基因组文库，该文库包含15600个克隆，外源片段DNA平均大小为18kb。通过DNA序列测定和同源性比较，对从文库中随机挑取的10个克隆进行了序列分析，发现9个外源插入片段包含未知DNA序列。

一种直接用于PCR的土壤微生物DNA提取方法

以橡胶林土壤为材料,直接提取土壤微生物DNA。本实验介绍的方法不仅可以获得大片段,并且不需要纯化即可直接用于PCR扩增和DNA酶切等后续操作,每克土壤DNA提取量约为2.1～4.6μg。此方法操作简单、快捷、为土壤宏基因组文库的构建和土壤微生物群落结构的多样性分析提供基础。

三种土壤微生物总DNA提取方法的比较

本文对3种常用的土壤微生物总DNA提取方法Martin法、高盐改进法及试剂盒法进行了比较,并通过DNA得率、纯度及16S rDNA V3可变区的PCR扩增结合DGGE法(denaturinggradient gel electrophoresis),分别对3种方法进行评价。结果表明,3种方法提取的DNA均能满足土壤微生物多样性分析的要求。其中试剂盒方法操作简单,提取的DNA质量较高,但DNA得率较低且成本昂贵。Martin法和高盐改进法用时较长,DNA得率较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。

红树林土壤总DNA不同提取方法比较研究

获得高浓度、大片段、无偏好的土壤微生物总DNA是土壤微生物分子生态学研究和宏基因组文库构建的基础。本研究采用了5种方法从红树林土壤中提取DNA,并对5种方法提取出的DNA的质量和产量进行比较评价。结果表明,5种方法均可从土壤中提取到DNA,但不同方法提取到DNA的产量和质量存在明显差异。Bio101FastPrep?SPINKit(forSoil)抽提到的DNA得率最高,适合分子生态学研究;SDS-GITC-PEG法提取的DNA纯度最高,所得到的DNA片段较大(>48kb),有利于构建宏基因组文库。

一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用PEG8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl2和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9·22μg·g-1,A260/A280为1·65,可适用于PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0·67ng·μl-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.