三种提取柱状黄杆菌基因组DNA方法的比较

采用酚氯仿抽提法、CTAB法和SDS-蛋白酶K法分别对鱼类病原菌柱状黄杆菌提取基因组DNA。使用超微量紫外分光光度计和琼脂糖凝胶电泳检测所提取的基因组DNA的产量和质量,并用PCR扩增对DNA进行了评价。结果显示,3种方法均可提取到柱状黄杆菌的基因组DNA,并能有效扩增细菌16S rDNA序列,但CTAB法提取的DNA产量和质量最高,CTAB法可以作为柱状黄杆菌DNA提取以开展分子生物学研究的首选方法。     

Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41 L. monocytogenes strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG), interacted with the DNA in a manner which restored adhesion. If a short DNA fragment (less than approximately 500 bp long) was added to an eDNA-free culture prior to addition of genomic or salmon sperm DNA, adhesion was prevented, indicating that high-molecular-weight DNA is required for adhesion and that the number of attachment sites on the cell surface can be saturated.The food-borne pathogen Listeria monocytogenes is known to persist in food processing plants (28, 48), and it has been reported that some strains of this species are capable of forming biofilms (2, 16). The mechanisms of biofilm formation have not been elucidated, but this process seems to depend on factors such as temperature and inducing compounds (14). One inducing compound is NaCl (22), but ethanol, isopropanol (14), quorum sensing (36), and an increasing temperature (8, 14, 38) also seem to enhance attachment and biofilm formation, whereas an acidic pH reduces adhesion (17, 38, 43). Furthermore, at 30°C flagellum-based motility seems to be a specific determinant for the initial adhesion (23, 42) and biofilm formation (23); however, it has recently been reported that in time nonflagellated mutants can produce hyperbiofilms (42).Since bacteria adhering to surfaces, both in biofilms and as single cells, exhibit increased resistance to sanitizers and antimicrobial agents (10, 41), examining the essential steps in adhesion and biofilm formation is important in order to develop new and improved sanitation processes.Extracellular DNA (eDNA) is a ubiquitous component of the organic matter pool in soil, marine, and freshwater habitats (26), but it is also found in environments as diverse as tissue cultures and the blood of mammals (11, 25). The presence of eDNA in the matrix of multicellular structures has recently been reported to influence the initial attachment and/or biofilm structure of Pseudomonas (1, 47), Streptococcus (29), and Staphylococcus (21, 33, 34) species.The prevalence of eDNA in nature appears to be associated with both lysis of cells and active secretion. The concentrations of eDNA released can be up to 2 μg g⁻¹ soil (30) and up to 0.5 g (m²)⁻¹ in the top few centimeters of deep-sea sediment (where more than 90% of the DNA is extracellular) (5). In the deep sea eDNA plays a key role in the ecosystem, functioning as a nitrogen and phosphorus reservoir (5). At present, there are different theories concerning both the function and the release of eDNA in multicellular structures. The presence of eDNA could be a result of either cell lysis (33, 34) or vesicle release (47), whereas active transport is a more speculative explanation. The role of eDNA in biofilm structure has not been revealed yet, but various functions, including a role as a structural component, an energy and nutrition source, or a gene pool for horizontal gene transfer (HGT) in naturally competent bacteria, can be envisaged.Until now there have been no studies of L. monocytogenes eDNA as a possible matrix component in relation to adhesion and biofilm development. In this study, we determined for the first time the presence of L. monocytogenes eDNA, its origin, and its role as a matrix component for both single-cell adhesion and biofilm formation using static assays, as well as flow cell systems. Furthermore, we showed that an additional component is necessary for eDNA-mediated adhesion.     

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The ¹³C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional ¹⁵N and ³¹P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.     

Chelex-100法及酚氯仿法提取阴道毛滴虫DNA的比较

目的-比较Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA。方法-分别用Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA,用PCR法检测DNA提取的有效性。结果两种方法提取的DNA经PCR扩增均有特定的条带。结论-两种方法均能提取阴道毛滴虫DNA。Chelex-100方法简便、省时,较适用于分子生物学研究及临床PCR扩增使用。     

Rapid Methods for Extracting Autolysins from Bacillus subtilis

Two procedures are described for the extraction of autolysins from whole cells. One method uses 5 M LiCl at 4 C. The amount of enzyme obtained by this method is six times more than that obtained by autolysis of cell walls and fourteen times more than that obtained by extracting cell walls with LiCl. With the other method, cells are extracted with 2% Triton X-100. This is less efficient than the LiCl method but yields about one-half the amount of enzyme obtained by cell wall autolysis and about the same amount as obtained by extracting cell walls with salt. Both procedures yield autolysin with multiple pH optima. Autolysins can be extracted from several bacterial species by either the LiCl or the detergent method. The data suggest that these techniques have sufficient sensitivity to detect small differences in autolytic activity among mutants and various organisms and are also suitable for large-scale isolation of autolysin for purification and characterization studies.     

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。     

獐不同组织材料DNA提取的有效方法

根据野外采集的獐肌肉、皮张、毛发、血迹、骨骼和粪便等不同样本的特点采用相适合的DNA提取方法,并对肌肉、皮张、毛发、骨骼的提取进行了改进,通过对线粒体细胞色素b和控制区基因PCR扩增反应以及测序结果证实,这几种DNA抽提方法及相应改进的可靠性,并可以提高野外非损伤性取材在保护遗传学中的应用。     

脂肪组织RNA提取方法的改进

本试验以猪脂肪组织为材料,应用TRIzol、V-gene、H.Q＆.Q三种总RNA提取试剂盒分别提取脂肪组织RNA,发现直接按照试剂盒说明书操作提取的RNA效果不理想.通过反复试验,分别改进了提取过程中的部分操作步骤,结果表明：三种试剂盒操作方法改进后与改进前相比,提取RNA的纯度和得率表现为差异极显著（P＜0.01）;三种试剂盒操作方法改进后提取RNA的纯度差异不显著（P＞0.05）,但在得率方面,Trizol比其它两种高,表现为差异极显著（P＜0.01）.三种试剂盒操作方法改进后提取的RNA经RT-PCR扩增猪β-actin基因,电泳结果显示,扩增条带清晰、特异,说明提取的RNA完全符合后续的分子生物学实验要求.     

三种提取冬虫夏草菌丝体基因组DNA方法的比较

采用改良氯化苄法、CTAB法、蛋白酶K裂解法这三种方法,对冬虫夏草菌丝体基因组DNA进行提取,将提取的基因组DNA经过紫外分光光度计检测纯度和计算浓度、琼脂糖凝胶电泳分析及PCR扩增,结果表明,3种方法均能提取到符合分子生物学的DNA,其中蛋白酶K裂解法纯度最高,含蛋白质少,改良氯化苄法、CTAB法次之。而浓度则是改良氯化苄法最高,CTAB法、蛋白酶K裂解法次之。3种方法所提取的DNA均能扩增出理想条带。     

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D₂O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.     

Giant Extracellular Matrix Binding Protein Expression in Staphylococcus epidermidis is Regulated by Biofilm Formation and Osmotic Pressure

Staphylococcus epidermidis is an opportunistic bacterium that thrives as a commensal cutaneous organism and as a vascular pathogen. The S. epidermidis extracellular matrix binding protein (Embp) has been reported to be a virulence factor involved in colonization of medical device implants and subsequent biofilm formation. Here, we characterize the expression patterns of Embp in planktonic and biofilm cultures, as well as under high osmotic stresses that typify the commensal environment of the skin. Embp expression without osmotic stress was similar for planktonic and adherent cultures. Addition of osmotic stress via NaCl caused slight increases in embp expression in planktonic cultures. However, in adherent cultures a 100-fold increase in embp expression with NaCl versus controls occurred and coincided with altered biofilm morphology. Results suggest that the central role of Embp lies in commensal skin colonization, stabilizing the cell wall against osmotic stresses, rather than as a virulence factor promoting adhesion.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Presence of Extracellular DNA in the <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Matrix and its Contribution to Biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.     

基质金属蛋白酶与血管壁细胞外基质重建

细胞外基质（ECM）不仅维持血管壁的完整性,而且还为血管细胞传递增殖,迁移,分化和凋亡的调控信号,基质金属蛋白酶（MMP）及其内源性抑制剂（TIMP）通过调节ECM合成与降解之间的动态平衡,使ECM维持正常的结构与功能。在高血压,动脉粥样硬化与血管再狭窄的发生与发展过程中,MMP和TIMP的合成与分泌出现异常,由此所引起的ECM合成与降解失调使ECM迅速发生重建。     

细胞外基质在植物发育中的作用

植物细胞壁是由纤维素和果胶交联的多糖和蛋白质构成的既彼此独立,又相互作用的三维动力学网络。和动物的细胞外基质一样,植物细胞壁中的许多成分积极地参与植物细胞发育过程的调节,它们以某种方式将信息传递给细胞,调节细胞的行为,以便对各种外界环境作出相应的反应。因此细胞壁不再是一种环绕植物细胞的惰性结构,比起细胞壁,植物细胞外基质这一名词更能反映出这一动力学的特性。     

微量法鼠疫菌质粒DNA抽提的优化

旨在分析微量法抽提鼠疫菌质粒DNA的效果,探讨其在鼠疫菌分子生物学实验研究中的应用价值.采用微量法分别提取鼠疫菌EV76株,假结核耶尔森菌PstII株及大肠杆菌V517株质粒DNA,琼脂糖凝胶电泳对质粒DNA抽提结果进行分析.结果显示,微量法能在较短时间内获取开环较少的闭合环状鼠疫菌质粒DNA,经琼脂糖凝胶电泳图示其电泳条带清晰、亮度均一.微量法鼠疫菌质粒DNA抽提效率和纯度较好,抽提结果稳定,重复性良好.经微量法抽提的质粒DNA符合多数鼠疫菌分子生物学试验的要求,可广泛应用于鼠疫菌分子生物学试验研究中.     

Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

Background

DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction.

Methodology/Principal Findings

From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol® reagent, Puregene® solutions and DNeasy® column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy® kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle''s genome, and all samples showed successful amplifications.

Conclusion/Significance

These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.