scholarly journals A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  
Keyword(s):  
Dna Extraction ◽  
Foodborne Pathogens ◽  
Extraction Method ◽  
Pathogenic Bacteria ◽  
Low Cost ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Bacterial Dna ◽  
Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

scholarly journals Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L.

OENO One ◽  
2013 ◽  
Vol 47 (4) ◽  
pp. 227 ◽  
Author(s):  
Gemma Marsal ◽  
Núria Boronat ◽  
Joan Miquel Canals ◽  
Fernando Zamora ◽  
Francesca Fort
Keyword(s):  
Vitis Vinifera ◽  
Dna Extraction ◽  
Woody Plants ◽  
Extraction Method ◽  
Low Cost ◽  
Handling Time ◽  
Extraction Methods ◽  
Original Method ◽  
Dna Extraction Method ◽  
Functional Dna

<p style="text-align: justify;"><strong>Aim</strong>: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of <em>Vitis vinifera</em> woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.</p><p style="text-align: justify;"><strong>Methods and results</strong>: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal <em>et al</em>. (2011) is proposed to reduce handling time and cost.</p><p style="text-align: justify;"><strong>Conclusion</strong>: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal <em>et al</em>. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.</p>

scholarly journals Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 19 ◽  
Author(s):  
Agata Wesolowska-Andersen ◽  
Martin Bahl ◽  
Vera Carvalho ◽  
Karsten Kristiansen ◽  
Thomas Sicheritz-Pontén ◽  
...  
Keyword(s):  
Community Structure ◽  
Dna Extraction ◽  
Extraction Method ◽  
Metagenomic Analysis ◽  
Fecal Material ◽  
Bacterial Dna ◽  
Dna Extraction Method ◽  
Bacterial Dna Extraction

scholarly journals Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

2020 ◽  
Vol 5 (2) ◽  
pp. 95 ◽  
Author(s):  
Rajashree Chowdhury ◽  
Prakash Ghosh ◽  
Md. Anik Ashfaq Khan ◽  
Faria Hossain ◽  
Khaledul Faisal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Recombinase Polymerase Amplification ◽  
National Guidelines ◽  
Diagnostic Efficacy ◽  
Rapid Extraction ◽  
Kala Azar ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

scholarly journals A modified SDS-based DNA extraction method from raw soybean

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Yimiao Xia ◽  
Fusheng Chen ◽  
Yan Du ◽  
Chen Liu ◽  
Guanhao Bu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Isoamyl Alcohol ◽  
Monitoring Methods ◽  
Detection Techniques ◽  
Dna Yield ◽  
Seed Flour ◽  
Dna Extraction Method ◽  
Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

scholarly journals Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

2019 ◽  
Author(s):  
Q.R. Ducarmon ◽  
B.V.H. Hornung ◽  
A.R. Geelen ◽  
E.J. Kuijper ◽  
R.D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACTWhen studying the microbiome using next generation sequencing, DNA extraction method, sequencing procedures and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing, and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with maximum three-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for estimating richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs and variation induced by DNA extraction methods was lower than inter-subject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we demonstrate the importance of including negative controls when analyzing low bacterial biomass samples.IMPORTANCEMethod choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls, and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiome studies. Our methods were not only applied to commonly studied samples for microbiota analysis, e.g. feces, but also for more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g. urine and tumor biopsies) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiome studies, especially when low bacterial biomass is expected.

eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

2021 ◽  
Author(s):  
Ana Laca ◽  
Abigail Wells ◽  
Linda Park
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Isoamyl Alcohol ◽  
Dna Purification ◽  
Phase Lock ◽  
Dna Extraction Method ◽  
Aqueous Layer

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

scholarly journals Fecal genomic DNA extraction method impacts outcome of MinION based metagenome profile of tuberculosis patients.

2021 ◽  
Author(s):  
Sukanya Sahu ◽  
Sandeep Kaushik ◽  
Bidhan Goswami ◽  
Arunabha Dasgupta ◽  
Hritusree Guha ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Dna Extraction ◽  
Gut Microbiome ◽  
Extraction Method ◽  
Critical Role ◽  
Isoamyl Alcohol ◽  
Fecal Samples ◽  
Sequencing Platform ◽  
Dna Extraction Method ◽  
Generation Sequencing

In the present era, emergence of next generation sequencing approaches has revolutionized the field of gut microbiome study. However, the adopted DNA extraction step used in metagenomics experiments and its efficiency may play a critical role in their reproducibility and outcome. In this study, fecal samples from active and non-tuberculosis subjects (ATB/NTB, n=7) were used. Fecal samples of a subgroup of these subjects were subjected to Mechanical enzymatic lysis (MEL) and Phenol: Chloroform: Isoamyl Alcohol (PCIA) methods of DNA extraction and a third-generation sequencing platform i.e., MinION was employed for microbiome profiling. Findings of this study demonstrated that DNA extraction method significantly impacts the DNA yield and microbial diversity. Irrespective of the adopted method of DNA extraction, ATB patients showed altered microbial diversity compared to NTB controls. Also, the fecal microbial diversity details are better captured in samples processed by MEL method and may be suitable to be adopted for high-throughput gut microbiome studies.

scholarly journals A low-cost pipeline for soil microbiome profiling

2020 ◽  
Author(s):  
Anita Bollmann-Giolai ◽  
Michael Giolai ◽  
Darren Heavens ◽  
Iain Macaulay ◽  
Jacob Malone ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Low Cost ◽  
Sequence Variants ◽  
Genomic Dna Extraction ◽  
Library Construction ◽  
Dna Extraction Method ◽  
Amplicon Library ◽  
Commercial Kits

AbstractBackgroundCommon bottlenecks in environmental microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in PCR inhibitors. To address this, we have established a low-cost genomic DNA extraction method for inhibitor rich samples alongside an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (2017). Our benchmarking experiment used four different soil types (coniferous, broad leafed, and mixed forest plus a standardised cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analysing sequence variants of 16S V4 and ITS rRNA amplicons.ResultsWe found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing delivered the cleanest genomic DNA e.g. best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits.ConclusionHere we present a low-cost genomic DNA extraction method for inhibitor rich sample types such as soil that can be coupled to an Illumina-compatible simple two step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large scale amplicon sequencing projects. Notably our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, Dovetail genomics etc.

scholarly journals Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria

2020 ◽  
Vol 48 (5) ◽  
pp. 030006052092559
Author(s):  
Xiaolan Li ◽  
Caroline J. Bosch-Tijhof ◽  
Xi Wei ◽  
Johannes J. de Soet ◽  
Wim Crielaard ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Efficiency ◽  
Bacterial Species ◽  
Gram Positive ◽  
Bacterial Dna ◽  
Bacterial Cultures ◽  
Dna Extraction Method ◽  
Mp Method ◽  
Bacterial Dna Extraction

Objective Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. Methods Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. Results For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. Conclusions The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

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