Comparison of five assays for DNA extraction from bacterial cells in human faecal samples

Aim To determine the most effective DNA extraction method for bacteria in faecal samples. Materials and Results This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. Conclusion From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. Significance and Impact of the Study The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

Background: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Methods: DNA was extracted from two RDT devices (Paracheck-PfW and SD Bioline Malaria Pf/Pan (R)), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman (R) 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. Results: The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. Conclusions: The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.ACT Consortium through Bill and Melinda Gates Foundation; Swedish International Development Agency (SIDA) [SWE 2009-193]; Swedish Civil Contingencies Agency (MSB) [2010-7991]; Swedish Medical Research Council (VR) [2009-3785]; Goljes Foundationinfo:eu-repo/semantics/publishedVersio

Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

BACKGROUND: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. METHODS: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. RESULTS: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots. CONCLUSION: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity

An efficient protocol to perform genetic traceability of tissue and foods from Geoffroea decorticans

The quality of a DNA isolation method depends, among others, on the target tissue and the metabolites therein. Geoffroea decorticans Burkart (chanar) is a species that has nutritional and pharmacological potential. However, an effective method of DNA extraction capable of facilitating population studies and food genetic traceability has not been studied yet. The objective of the present work was to evaluate four methods of DNA extraction from leaves and chanar-based foods. The methods were evaluated based on yield, DNA purity, and molecular markers. The CCI-P (CTAB/Chloroform-Isoamylalcohol/pellet) method showed the highest yield of DNA obtained from leaves. However, the CPCI-SC (CTAB/Phenol-Chloroform-Isoamylalcohol/silica-column) method was the only one that resulted in acceptable DNA quality with both parameters (A260/A280 and A260/A230). The leaf DNA obtained with this method showed a greater amount of fragments with RAPD, and an acceptable amount of fragments with ISSR. On the other hand, the CCI-P method showed a higher yield of DNA from arrope de chanar (syrup). However, the CPCI-SC method was the only one that had relatively better DNA quality, which allowed the amplification of molecular markers. Regarding chanar flour, the CPCI-SC method showed the highest yield, DNA quality and good amplification with molecular markers. Therefore, the CPCI-SC extraction method is efficient for obtaining DNA from different matrices, and can support studies for a possible designation of origin of chanar-based foods

Rapid plant DNA and RNA extraction protocol using a bench drill

Plant DNA and RNA extraction methods are well established, with a wide range of protocols, depending on the purposes of each laboratory/research. Nowadays, quick, inexpensive and easy plant DNA and RNA extraction methods are highly sought after. We developed an optimized protocol for plant DNA and RNA extraction that uses an inexpensive bench drill and plastic bags and does not require liquid nitrogen. DNA from leaves and RNA from leaves and roots of banana, pineapple, citrus, papaya, passion fruit and cassava, were extracted using a basic cetyltrimethylammonium bromide method. Both nucleic acids were quantified and evaluated for quality based on agarose gel electrophoresis. The DNA and RNA extractions were successful for all species, and RNA quality in pellets was maintained after storage at room temperature for three weeks. This protocol can reduce costs considerably in laboratories with ongoing routine activities of DNA and RNA extraction for genetic diversity and gene expression analyses, where other conventional methods have not been successful due to explant, condition of samples and quantity and quality of nucleic acids. This is especially relevant for many laboratories in developing countries where the cost and availability of liquid nitrogen may be a constraint

Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Point of Care Diagnosis of Multiple Schistosome Parasites: Species-specific DNA Detection in Urine by Loop-mediated Isothermal Amplification (LAMP)

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% − 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis

A rapid and low-cost DNA extraction method for isolating Escherichia coli DNA from animal stools

The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA from goat, chicken, pig, cow and human stool samples. Two versions of the lysis buffer, with and without _-casein, were tested to alleviate PCR inhibition associated with DNA isolated from stool samples. Results obtained show that, this method using the lysis buffer containing _-casein, produces PCR ready DNA at a fraction of the cost of commercial DNA extraction kits & copy; 2011 Academic Journals

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells