A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

Background

Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell.

Principal Findings

Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample.

Conclusions

The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.     

Improving the recovery of qPCR-grade DNA from sludge and sediment

DNA extraction is often considered as the limiting step of most molecular approaches in ecology and environmental microbiology. Ten existing DNA extraction protocols were compared for recovery of DNA from sludge and a modified version of the protocol described by Porteous et al. (Mol Ecol 6:787–791, 1997) was determined to be the best method for recovery of DNA suitable for PCR. In this respect, it appeared that the commonly used guanidine isothiocyanate could impair the quality of the extracted DNA unless its concentration is lowered. Second, conditioning the samples as liquors as opposed to pellets critically impacts the outcome of the extraction. The suitability of the modified Porteous protocol for quantitative PCR applications is demonstrated in a series of experiments showing the absence of interfering coextracted inhibitors and the linear correspondence between the concentrations of input target DNA and PCR product. Interestingly, it is also shown that the nature of the environmental matrices affects the recovery yield of both circular plasmids and chromosomal DNA, resulting in an apparent fluctuation of the plasmid copy number per cell. This means that quantitative data obtained by PCR remain comparable as long as they apply to an identical target sequence extracted from a similar environment and amplified under the same conditions.     

A method for the medium-term storage of plant tissue samples at room temperature and successive cycles of DNA extraction

Based on the protocol originally described by Stein et al. (2001), we have developed a method that allows for medium-term conservation at room temperature of wheat (Triticum aestivum) tissue samples to use for DNA extraction. DNA quality was suitable for analysis by PCR and Southern hybridization, even after 2 months of storage at room temperature. This method allows successive DNA re-extractions from a previously extracted sample and maximization of the DNA yield that can be recovered from precious samples. This method has applications for conservation of leaf samples and management of DNA extraction. Our method can help improve data recovery in many plant molecular genetics research projects.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

DNA extraction from bronchial aspirates for molecular cytology: which method to take?

OBJECTIVE: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods. METHODS: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated. RESULTS: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of beta-globin gene fragments (268, 536, and 989 bp) was 100%. CONCLUSION: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR-based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.     

鳞翅目成虫乙醇保存标本基因组DNA的提取及在微卫星研究中的应用

高质量的基因组DNA样品是分子生态学研究的先决条件。本研究目的在于探索从东方粘虫Pseudaletia separata (Walker)成虫自然种群的乙醇保存标本中分离高质量基因组DNA的有效方案。在2 mL微型离心管中进行4种提取方案的实验比较,结果发现采用传统的苯酚抽提方法的2种方案提取腹部中段组织的基因组DNA,样品合格率只有7.69%~40%。但是,如果在苯酚抽提以前加入高浓度盐和十六烷基三甲基溴化铵（CTAB）,就会使DNA样品合格率达到68.42%~95.28%,而且DNA平均产量达到5.59~10.04 mg/g,明显高于前者的2.83~5.78 mg/g （统计检验表明,在不同种群中差异显著或不显著）。研究结果还证明腹部组织比胸部组织更适宜提取DNA。对来自一个自然种群的99头东方粘虫DNA合格样品的统计分析表明,DNA提取总量（μg）与组织样品用量（mg）之间存在弱的正相关关系,平均DNA提取量（mg/g）与组织样品用量（mg）之间存在中度负相关关系。总之,在2 mL微型离心管中,用10~20 mg腹部组织,利用CTAB+苯酚抽提方法可以获得高纯度和高含量的基因组DNA样品。用该方案提取的基因组DNA能够顺利地进行微卫星位点的分离和基因分型。     

Assessment of various methods for extraction of metagenomic DNA from saline habitats of coastal Gujarat (India) to explore molecular diversity

Aims:  To develop total DNA extraction protocol from saline soil for further metagenomic applications.
Methods and Results:  The protocols combine the application of mechanical (Beads and Sonicator) and Soft Lysis (SDS and enzymes) method for the isolation of total DNA from saline soil of coastal Gujarat followed by its quantification and purity assessment. The quality and purity of metagenomic DNA was quite consistent and reliable, although it contained residual concentartions of humic acid. The extracted DNA was used to successfully amplify 16S rRNA region. The amplicons were suitable for further applications such as diversity-based analysis by denaturing gradient gel electrophoresis (DGGE).
Conclusions:  The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA.
Significance and Impact of the Study:  The protocol is simple, short and facilitates rapid isolation of PCR amplifiable total genomic DNA from saline soil. The method yielded good quality of the DNA suitable for metagenomic studies. The results are also significant as only few extreme environments, particularly saline habitats, are explored for their metagenomic potential.     

DNA extraction method for PCR in mycorrhizal fungi

AIMS: To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. METHODS AND RESULTS: The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. CONCLUSION: DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.     

Extraction and detection of baculoviral DNA from lake water, detritus and forest litter

AIMS: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. METHODS AND RESULTS: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt-/lacZ+. The detection limit of PCR amplification was routinely about 4.1 x 102 occlusion bodies (OBs) 450 microl-1 lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 103 and 2.08 x 104 OBs 0.5 g-1 dry weight, respectively. CONCLUSION: The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.     

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.     

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased,and cost-effective method for the isolation of high molecular weight(23 kb) metagenomic DNA(260/280 ratio 1.8) with a good yield(55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination(eubacterial: human genomic DNA marker genes = 2~(27.9):1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16 S r RNA gene sequencing using 454 FLX+.Moreover, 16 S r RNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richnessand does not show microbial diversity biases, which is further confirmed by q PCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality,amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.     

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.     

Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications

We evaluated the robustness of loop-mediated isothermal amplification (LAMP) of DNA for bacterial diagnostic applications. Salmonella enterica serovar Typhi was used as the target organism and compared with a real-time quantitative PCR (qPCR) for testing assay performance and reproducibly, as well as the impact of pH and temperature stability. This isothermal amplification method appeared to be particularly robust across 2 pH units (7.3-9.3) and temperature values (57-67 °C). The detection limit was comparable to that observed using optimized home-brew qPCR assays. The specificity of the amplification reaction remained high even at temperatures markedly different from the optimal one. Exposing reagents to the ambient temperature during the preparation of the reaction mixture as well as prolonging times for preparing the amplification reaction did not yield false-positive results. LAMP remained sensitive and specific despite the addition of untreated biological fluids such as stool or urine that commonly inhibit PCR amplification. Whereas the detection of microorganisms from whole blood or a blood-culture medium typically requires extensive sample purification and removal of inhibitors, LAMP amplification remained more sensitive than conventional qPCR when omitting such preparatory steps. Our results demonstrate that LAMP is not only easy to use, but is also a very robust, innovative and powerful molecular diagnostic method for both industrialized and developing countries.     

一种快速、简单的琼脂糖凝胶DNA片段分离法

该方法利用Ｎａｌ溶解凝胶,硅胶颗粒吸附ＤＮＡ片段便之分离。有快速、不影响后续酶反应、高回收率等特点,可用于基因工程中酶切片段、ＰＣＲ产物的分离纯化。     

一种提取质粒DNA的改良方法

本文详细介绍了一种改良碱裂解法提取质粒ＤＮＡ的方法,该法采用ＮＨ４Ａｃ代替苯酚和氯份的抽提过程,得率高,质量好,完全达到了分子生物学常规实验的要求,如酶切、连接、转化大肠杆菌、ＰＣＲ等,甚至用于序列测定和植物遗传转化,该法重复性好,操作简单、实用．     

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.     

DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

Real-time PCR to study the sequence specific magnetic purification of DNA

The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations.