四种小鼠肠道微生物DNA提取方法比较

采用异硫氰酸胍(guandine thiocyanate,GITC)法、Tiangen DNA提取试剂盒、Omega DNA提取试剂盒和广泛应用的十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)法提取小鼠粪便微生物总DNA,通过比较所提取DNA的浓度和纯度,发现粪便DNA提取试剂盒提取的DNA纯度最高但浓度最低;CTAB法所得的DNA浓度最高但纯度最低;GITC法所得DNA的浓度高于粪便DNA提取试剂盒,纯度高于CTAB法。通过变性梯度凝胶电泳(16S r DNA-PCR-DGGE)指纹图谱分析技术进一步比较了各种提取方法所代表微生物群落的丰富度和多样性。结果表明,GITC法提取得到的DNA所代表细菌的丰富度和多样性显著高于其他3种方法。本实验所建立的GITC法可更全面地反映肠道微生物的多样性和群落结构,是一种较为理想的粪便微生物DNA提取方法。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的：在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法：分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果：相同处理量（108个）的革兰氏阳性菌（1株）、革兰氏阴性菌（4株）、古菌（1株）经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260／OD280的值更接近1．8-2．0（纯DNA的OD260／OD280在1．8—2．0之间）,前者所提DNA回收率最大可达后者的16．7倍;煮沸裂解法只需较少菌（102个）便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论：两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随茵浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

湖泊沉积物基因组DNA的提取方法比较研究

水体沉积物为一新型微生物种质资源库,不依赖于培养的菌种多样性研究的首要条件是获取优质的基因组DNA。本研究以改进的氯仿异戊醇法、蛋白酶K+SDS法、玻璃珠+蛋白酶K+SDS法提取湖泊基因组DNA,并与试剂盒提取结果进行比较,为沉积物分子生态学的研究提供参考和借鉴。结果表明,改进后的氯仿异戊醇法提取的DNA纯度较低,腐殖质明显,纯化后PCR产量较低;蛋白酶K+SDS法获得的DNA完整性较好,浓度高,PCR扩增应用效果最好;玻璃珠+蛋白酶K+SDS法提取的基因组DNA效果次之;试剂盒法提取的基因组DNA浓度较低,片段长度略小。蛋白酶K+SDS法为除试剂盒外适合于湖泊沉积物基因组DNA提取的较好方法。     

快速提取肠道微生物基因组DNA的方法

目的:肠道微生物的研究日益成为热点,如何获取高质量、较完整的肠道菌群基因组DNA是肠道微生物研究中的关键。本文通过对酚/氯仿法提取总DNA过程进行考察和优化,建立一种简便酚/氯仿抽提法。方法:考察和优化酚/氯仿法提取总DNA的过程,并根据DNA产量、纯度以及ERIC-PCR及16S rNDA-RFLP所反映的微生物群落结构特性的指标,并与QIAamp?DNA Stool Mini Kit提取的进行比较,评价了所建立的快速提取方法。结果:用简便酚/氯仿法得到基本完整的基因组DNA,ERIC-PCR和16S rDNA-RFLP结果与QIAamp?DNA Stool Mini Kit法基本相同。结论:该方法快速并成本低,适合肠道微生物研究中总DNA提取,尤其适合处理大批量的样品。     

一种快速经济提取革兰氏阴性和革兰氏阳性细菌基因组DNA的方法

细菌基因组DNA提取是分子生物学技术应用的基础,提取的DNA的质量可直接影响其结果的准确性和精确性。本研究提出了一种快速、经济的适用于多种细菌的基因组DNA提取方法,并比较了本方法与传统酚-氯仿法和试剂盒法提取3种革兰氏阴性细菌和4种革兰氏阳性细菌DNA的效果。结果显示,3种方法均可提取到较完整的基因组DNA(约23 kb)。在DNA纯度上,本法与传统酚-氯仿法无显著差异,略低于试剂盒法,但可满足PCR扩增等分子生物学技术的要求,并且其提取效率高于试剂盒法。在成本方面,本法单个样本的成本仅为试剂盒法的20%,且完成整个实验的时间仅为传统酚-氯仿法和试剂盒的50%(约60 min)。总之,本研究提出了一种快速、经济、适用于G-和G+细菌的基因组DNA提取方法,本法不仅适用于本研究中的各种细菌,对其它革兰氏阴性细菌和革兰氏阳性细菌也具有重要的潜在应用价值。     

鸡肠道菌群总DNA三种提取方法的比较

目的比较不同方法提取鸡肠道菌群总DNA的差异,为分子方法分析肠道菌群组成提供质量较高的DNA模板。方法采用反复冻融法、酶裂解法和试剂盒法（E.N.Z.A Stool DNA Kit）来提取鸡肠道菌群的总DNA,并根据DNA浓度及纯度、16S DNA扩增产物和ERIC-PCR产物所反映的片段多态性4个指标,对这3种方法提取的DNA质量进行比较。结果3种方法均能提取DNA,所得DNA都可以用于16S DNA的扩增,但后2种方法所得DNA的ERIC-PCR结果能反映出更高的菌群多样性。结论试剂盒法和酶裂解法所提取的DNA质量好,适合用于肠道菌群的分子生态研究。     

一种从活性污泥中提取微生物总DNA的方法

对活性污泥的微生物群落进行研究的首要前提是获得大量的高纯度微生物基因组DNA。本文建立了一种高效、简便的提取活性污泥总DNA方法。从提取的核酸总量、纯度、基因组完整性等多方面对所得到的DNA质量进行了评价,结果表明,本法从单位活性污泥中提取的DNA得率为105-823μg/g,结构完整,纯度很高,无需进一步的纯化,可直接进行微生物群落分析及构建文库等后续分子生物学操作。现在实验室使用的提取活性污泥中DNA的方法,纯度普遍都无法达到PCR反应和建立文库的要求,本文建立的活性污泥DNA提取方法则可以克服这一难题。     

短序十大功劳基因组总DNA提取方法研究

以短序大功劳嫩叶为材料,采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率,用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明,五种方法均能从短序大功劳叶片中提取到基因组DNA,但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高,可直接用于下游分子生物学实验,CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差,不利于下游的分子生物学实验;五种方法提取的总DNA的得率在10.836~451.709μg/g之间,呈CTAB法>SDS法>CTAB改良法1>CTAB改良法2>试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。     

小鼠胚胎胃肠道细菌基因组DNA提取方法比较

目的:筛选能均衡地提取小鼠胚胎胃肠道微生物区系各种细菌总DNA的方法.方法:分别采用反复冻融法、CTAB -SDS法、柱式基因组DNA提取试剂盒法提取小鼠胚胎胃肠道细菌基因组DNA,对其进行琼脂糖凝胶电泳、紫外分光光度计测定、PCR扩增等质量检测.结果:CTAB -SDS方法提取的基因组DNA纯度较高,OD260/OD280平均值最高,为1.845,电泳条带清晰,能满足下游的PCR扩增等分子操作.结论:确定CTAB -SDS方法为提取小鼠胚胎胃肠道细菌基因组DNA的最佳方法,为研究不同种动物胚胎的肠道菌群的结构和多样性奠定了基础.     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA^@ SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae.     

A comparison of five methods for extraction of bacterial DNA from human faecal samples

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.     

Efficient isolation method for high‐quality genomic DNA from cicada exuviae

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

Bacterial and Archaeal DNA Extracted from Inoculated Experiments: Implication for the Optimization of DNA Extraction from Deep-Sea Basalts

Difficulties in efficient DNA extraction from deep-sea volcanic basalt, due to high metal concentration, complex organic matter, or sometimes the low biomass, have hampered the understanding of the significant biosphere both at and below the sea floor. In order to optimize the DNA extraction from basaltic rocks, sterilized basalts with different particle sizes and chemically synthesized goethite were inoculated with an iron oxidizer Marinobacter aquaeolei and an extreme halophilic archaeon Halobaculum gomorrense respectively, and extracted with several methods. Large variations in DNA yields by different extracting methods including FastDNA® spin for soil kit, GeneClean® for ancient DNA kit, UltraClean? and traditional phenol-chloroform methods. Among the commercially available kits tested here, FAST spin kit and GeneClean® for ancient DNA kit yield 10 times more DNA than the UltraClean? kit used. In combination with FAST spin kit, skim milk greatly enhanced the archaeal DNA yields. DNA extracting efficiency was low with the cell number lower than 1 × 10⁶ cells, but reached as high as 88% with a cell number of 1 × 10⁸ cells. On these points, different strategies should be taken into consideration for the DNA extraction from basalts, depending on original biomass and cell types anticipated. FAST spin kit could provide high quality bacterial DNA for downstream PCR whilst the combination of FAST spin kit with skim milk would greatly enhance the archaeal DNA yields. GeneClean® for ancient DNA kit is also recommended for archaeal DNA extraction from deep sea basalt due to its high yield.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

革兰氏阳性细菌基因组DNA提取方法的比较及优化

【目的】基因组DNA提取效率和质量对分子生物学相关研究起着关键的作用,革兰氏阳性细菌由于细胞壁较厚、难破裂使其基因组DNA提取的难度增大,本文旨在寻找一种高效稳定的DNA提取方法。【方法】以Clostridium thermocellum和Thermoanaerobacterium thermosaccharolyticum为实验菌株,使用6种DNA提取方法对C. thermocellum基因组DNA进行提取,对比其提取效果和产率。【结果】改良的SDS-碱裂解法提取得到的DNA浓度较高(400 mg/l左右),且平行样间浓度和纯度稳定。【结论】为革兰氏阳性细菌基因组DNA提取提供参考。