Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.     

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.     

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.     

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (101 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 102 spores and 75 % for 101 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.     

Isolation of RNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.     

Isolation of DNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take approximately 2.5-4 hours.     

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.     

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations > 0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n = 24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.     

不同粪便DNA提取方法比较分析北大核心CSCD

肠道微生物群落结构和多样性与人体疾病密切相关。然而,相关群落结构分析结果可能受到DNA提取质量等实验因素影响。因此,评估不同DNA提取方法对肠道特定种属的提取效果,对于全面、准确获取人体肠道微生物谱,深入探究肠道微生物群落结构具有指导意义。本研究旨在借助实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT qPCR)技术,以DNA提取纯度、浓度,以及对肠道中特定种属微生物基因组DNA的提取丰度为指标,对5种DNA提取方法进行比较分析。结果表明,试剂盒Q的提取效果最佳,特别是对乳杆菌属和双歧杆菌属等革兰氏阳性菌的提取效果较好。N试剂盒的平均DNA提取浓度较Q试剂盒低,但在纯度方面,二者无显著性差异。与其他3种商用试剂盒(M、PSP、TG)相比,N方法对肠道内指定微生物基因组的提取效果仅次于Q试剂盒,位居第二。相比之下,M试剂盒提取所得DNA,质量较高,但浓度偏低,对于肠道内革兰氏阳性菌的提取效果不很理想。TG试剂盒和PSP试剂盒提取所得DNA在浓度、质量以及细菌丰度方面均不及其他验证的试剂盒。综上,Q试剂盒可作为肠道微生态研究相关实验中获取高质量基因组DNA的提取方法。本研究结果为肠道微生态研究相关实验中基因组DNA提取方法的选择提供参考依据。     

体重差异双胎新生儿粪便样本DNA提取的优化及分析

目的 优化新生儿粪便样本DNA提取方法,提取及分析体重差异双胎新生儿粪便样本DNA。方法 从7种DNA提取试剂盒方法中选择对成人粪便样本DNA提取效果最佳的QIAamp DNA Stool Mini Kit法为基准方法,通过钢珠打断前处理、DNA吸附柱收集全部裂解液上清和洗脱液重复洗脱的优化,建立了用于新生儿粪便样本DNA的提取方法。结果 该优化方法用于婴儿（出生1个月）粪便样本,结果显示DNA提取浓度平均提高了2.4倍。用于48对双胞胎新生儿出生第1天和第3天粪便样本DNA的提取,经酶标仪及PCR扩增检测,结果显示出生第1天粪便样本DNA提取率为32%,出生3天提取率达83%。RT-PCR显示新生儿第1天到第3天肠道微生物量呈现增长趋势。结论 优化的QIAamp DNA Stool Mini Kit法适用于新生儿粪便样本DNA的快速提取,为后续扩增子高通量测序和研究体重差异双胎新生儿肠道菌群构成规律奠定基础。     

Comparisons of direct extraction methods of microbial DNA from different paddy soils

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.     

FDA 2014 survey of eye area cosmetics for microbiological safety

The aim of this work was to evaluate a fungal DNA extraction procedure with the lowest inputs in terms of time as well as of expensive and toxic chemicals, but able to consistently produce genomic DNA of good quality for PCR purposes. Two types of fungal biological material were tested ‐ mycelium and conidia ‐ combined with two protocols for DNA extraction using Sodium Dodecyl Sulphate (SDS) and Cetyl Trimethyl Ammonium Bromide as extraction buffers and glass beads for mechanical disruption of cell walls. Our results showed that conidia and SDS buffer was the combination that lead to the best DNA quality and yield, with the lowest variation between samples. This study clearly demonstrates that it is possible to obtain high yield and pure DNA from pigmented conidia without the use of strong cell disrupting procedures and of toxic reagents.

Significance and Impact of the Study

There are numerous methods for DNA extraction from fungi. Some rely on expensive commercial kits and/or equipments, unavailable for many laboratories, or make use of toxic chemicals such as chloroform, phenol and mercaptoethanol. This study clearly demonstrates that it is possible to obtain high yields of pure DNA from pigmented conidia without the use of strong and expensive cell disrupting procedures and of toxic reagents. The method herein described is simultaneously inexpensive and adequate to DNA extraction from several different types of fungi.     

Comparison of Methods for Isolation of Bacterial and Fungal DNA from Human Blood

The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray

A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations (r=0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales, whereas a vigorous disruption produced peak intensities for Vibrionales, Clostridiales, and Bacillales.     

Microbiome of fungus-growing termites: a new reservoir for lignocellulase genes

Fungus-growing termites play an important role in lignocellulose degradation and carbon mineralization in tropical and subtropical regions, but the degradation potentiality of their gut microbiota has long been neglected. The high quality and quantity of intestinal microbial DNA are indispensable for exploring new cellulose genes from termites by function-based screening. Here, using a refined intestinal microbial DNA extraction method followed by multiple-displacement amplification (MDA), a fosmid library was constructed from the total microbial DNA isolated from the gut of a termite growing in fungi. Functional screening for endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase resulted in 12 β-glucosidase-positive clones and one xylanase-positive clone. The sequencing result of the xylanase-positive clone revealed an 1,818-bp open reading frame (ORF) encoding a 64.5-kDa multidomain endo-1,4-β-xylanase, designated Xyl6E7, which consisted of an N-terminal GH11 family catalytic domain, a CBM_4_9 domain, and a Listeria-Bacteroides repeat domain. Xyl6E7 was a highly active, substrate-specific, and endo-acting alkaline xylanase with considerably wide pH tolerance and stability but extremely low thermostability.     

Inducible cell lysis systems in microbial production of bio-based chemicals

The release of products from microbial cells is an essential process for industrial scale production of bio-based chemicals. However, traditional methods of cell lysis, e.g., mechanical disruption, chemical solvent extraction, and immobilized enzyme degradation, account for a large share of the total production cost. Thus, an efficient cell lysis system is required to lower the cost. This review has focused on our current knowledge of two cell lysis systems, bacteriophage holin–endolysin system, and lipid enzyme hydrolysis system. These systems are controlled by conditionally inducible regulatory apparatus and applied in microbial production of fatty acids and polyhydroxyalkanoates. Moreover, toxin–antitoxin system is also suggested as alternative for its potential applications in cell lysis. Compared with traditional methods of cell disruption, the inducible cell lysis systems are more economically feasible and easier to control and show a promising perspective in industrial production of bio-based chemicals.     

Technical difficulties and solutions of direct transesterification process of microbial oil for biodiesel synthesis

Microbial oils are considered as alternative to vegetable oils or animal fats as biodiesel feedstock. Microalgae and oleaginous yeast are the main candidates of microbial oil producers’ community. However, biodiesel synthesis from these sources is associated with high cost and process complexity. The traditional transesterification method includes several steps such as biomass drying, cell disruption, oil extraction and solvent recovery. Therefore, direct transesterification or in situ transesterification, which combines all the steps in a single reactor, has been suggested to make the process cost effective. Nevertheless, the process is not applicable for large-scale biodiesel production having some difficulties such as high water content of biomass that makes the reaction rate slower and hurdles of cell disruption makes the efficiency of oil extraction lower. Additionally, it requires high heating energy in the solvent extraction and recovery stage. To resolve these difficulties, this review suggests the application of antimicrobial peptides and high electric fields to foster the microbial cell wall disruption.     

Evaluation of the ISO Standard 11063 DNA Extraction Procedure for Assessing Soil Microbial Abundance and Community Structure

Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.