scholarly journals Evaluation of Extraction Methods for Clinical Metagenomic Assay

2020 ◽  
Vol 8 (8) ◽  
pp. 1128
Author(s):  
Suha A. Farraj ◽  
Shreif A. El-Kafrawy ◽  
Taha A. Kumosani ◽  
Jehad M. Yousef ◽  
Esam I. Azhar
Keyword(s):  
Nucleic Acid ◽  
Comparative Evaluation ◽  
Extraction Method ◽  
Extraction Methods ◽  
Multiple Infections ◽  
Infectious Agents ◽  
Nucleic Acid Isolation ◽  
Efficient Detection ◽  
Different Types ◽  
Etiological Agents

(1) Background: Clinical metagenomics is a promising approach that helps to identify etiological agents in cases of unknown infections. For the efficient detection of an unknown pathogen, the extraction method must be carefully selected for the maximum recovery of nucleic acid from different microorganisms. The aim of this study was to evaluate different extraction methods that have the ability to isolate nucleic acids from different types of pathogens with good quality and quantity for efficient use in clinical metagenomic identification. (2) Methods: A mock sample spiked with five different pathogens was used for the comparative evaluation of different commercial extraction kits. Extracted samples were subjected to library preparation and run on MiSeq. The selected extraction method based on the outcome of the comparative evaluation was used subsequently for the nucleic acid isolation of all infectious agents in clinical respiratory samples with multiple infections. (3) Results: The protocol using the PowerViral® Environmental RNA-DNA Isolation Kit with a 5-min bead beating step achieved the best results with a low starting volume. The analysis of the tested clinical specimens showed the ability to successfully identify different types of pathogens. (4) Conclusions: The optimized extraction protocol in this study is recommended for clinical metagenomics application in specimens with multiple infections from different taxa.

scholarly journals Evaluation of Biochar Nitrate Extraction Methods

2019 ◽  
Vol 9 (17) ◽  
pp. 3514 ◽  
Author(s):  
Jenna Walsh ◽  
Joseph Sanford ◽  
Rebecca Larson
Keyword(s):  
Extraction Method ◽  
Extraction Efficiency ◽  
Agricultural Soils ◽  
Extraction Methods ◽  
Multiple Methods ◽  
Soil Extraction ◽  
Future Studies ◽  
Different Types ◽  
Increasing Temperature ◽  
Biochar Amendment

Biochar amendment to soil is a method used to mitigate losses of nitrogen leaching through agricultural soils. Multiple methods for extraction of nitrogen have been used, and recent studies have indicated that traditional soil extraction methods underestimate biochar nitrate. This study evaluated the nitrate extraction efficiency of a KCl extraction method under different temperature (20 and 50 °C) and duration (24 and 96 h) conditions. Increasing the duration of extraction from 24 to 96 h did not have a significant impact on extraction efficiency. However, increasing temperature resulted in nitrate extraction efficiencies above 90%. Rinsing the biochar once with deionized (DI) water following filtration after extraction increased the extraction efficiency significantly, but any subsequent rinses were not significant. This study recommends extracting nitrate from biochar using 2 M KCl at 50 °C for a period of 24 h with one additional rinse to increase nitrate recovery above 90%. However, future studies should evaluate this procedure for different types of biochar produced from alternative biomasses and at varying temperatures.

scholarly journals Comparative Evaluation of Essential Oils of Helichrysum petiolare Hilliard & B.L. Burtt Obtained from Solvent-Free Microwave and Hydrodistillation Extraction Methods

2020 ◽  
Vol 32 (12) ◽  
pp. 3227-3238
Author(s):  
A.E. Aladejana ◽  
G. Bradley ◽  
A.J. Afolayan
Keyword(s):  
Essential Oil ◽  
Essential Oils ◽  
Comparative Evaluation ◽  
Extraction Method ◽  
Chemical Constituents ◽  
Extraction Methods ◽  
Solvent Free ◽  
Microwave Extraction ◽  
Deep Yellow ◽  
Method Of Extraction

Till date, no study has ever looked into the effect of extraction method on the chemical constituents of the essential oil of Helichrysum petiolare. This study therefore evaluated the effect of hydrodistillation and solvent free microwave extraction (SFME) methods on the chemical constituents of H. petiolare derived essential oils. The SFME derived essential oil was deep yellow and of higher yield than the pale yellow essential oil obtained through the hydrodistillation. There were substantial amounts of monoterpenes, monoterpene alcohols, sesquiterpenes, and sesquiterpene alcohols in both essential oils obtained. The SFME derived essential oil had 62 compounds as against the 52 derived through hydrodistillation. The SFME derived essential oil can therefore be suggested to be of better quality than of the hydrodistillation method. The compounds obtained in the essential oils have high pharmaceutical and cosmetic values, and as observed in this study, their quantity is dependent on method of extraction.

scholarly journals Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Nasir Ali ◽  
Rita de Cássia Pontello Rampazzo ◽  
Alexandre Dias Tavares Costa ◽  
Marco Aurelio Krieger
Keyword(s):  
Nucleic Acid ◽  
Extraction Method ◽  
Point Of Care ◽  
Extraction Methods ◽  
Vital Role ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Advantages And Disadvantages ◽  
Point Of Care Diagnostics ◽  
Working Principle

Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at thepoint of careorpoint of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system.

scholarly journals Comparative evaluation of commercially available manual and automated nucleic acid extraction methods for rotavirus RNA detection in stools

2013 ◽  
Vol 194 (1-2) ◽  
pp. 242-249 ◽  
Author(s):  
Mathew D. Esona ◽  
Sharla McDonald ◽  
Shifaq Kamili ◽  
Tara Kerin ◽  
Rashi Gautam ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Evaluation ◽  
Extraction Methods ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Rna Detection

scholarly journals Comparative Evaluation of Protease Extraction from Leaves of Syzygium polyanthum

2019 ◽  
Vol 19 (2) ◽  
pp. 503 ◽  
Author(s):  
Noorhafiza Yahya ◽  
Zatul Iffah Mohd Arshad ◽  
Siti Kholijah Abdul Mudalip ◽  
Siti Zubaidah Sulaiman ◽  
Rohaida Che Man ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Phosphate Buffer ◽  
Comparative Evaluation ◽  
Extraction Method ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Extraction Methods ◽  
Hydrogen Bond Acceptor ◽  
Sodium Phosphate Buffer

In this study, the comparison of four extraction methods for protease from the leaves of Syzygium polyanthum was evaluated. The methods include sodium phosphate buffer, 1-methyl-8-oxyquinolinium betaine (QB) solvent based extraction, hydrogen-bond acceptor (HBA) solvent based extraction and Trichloroacetic-acetone (TCA) precipitation. The effectiveness of these extraction methods was measured based on the specific activity demonstrated by the crude protease extracted in this experiment. The results indicated that QB solvent-based extraction method could extract the crude protease from the leaves of S. polyanthum with the highest specific activity of 10.204 ± 0.329 U/mg compared to other extraction methods.

scholarly journals Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

2016 ◽  
Vol 82 (9) ◽  
pp. 2595-2607 ◽  
Author(s):  
Pia K. Wüst ◽  
Heiko Nacke ◽  
Kristin Kaiser ◽  
Sven Marhan ◽  
Johannes Sikorski ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Extraction Method ◽  
Soil Microbial Communities ◽  
Extraction Methods ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Extraction ◽  
Content Type ◽  
Bacterial Ribosome

ABSTRACTModern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particularActinobacteriaandProteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms.

scholarly journals Performance Evaluation of Manual and Automated (MagNA Pure) Nucleic Acid Isolation in HPV Detection and Genotyping Using Roche Linear Array HPV Test

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Aikaterini Chranioti ◽  
Evangelia Aga ◽  
Niki Margari ◽  
Christine Kottaridi ◽  
Asimakis Pappas ◽  
...  
Keyword(s):  
Extraction Method ◽  
Area Under Curve ◽  
Linear Array ◽  
Multiple Infections ◽  
Predictive Values ◽  
Pure System ◽  
Nucleic Acid Isolation ◽  
Hpv Test ◽  
Roche Linear Array ◽  
Manual Extraction

Nucleic acids of human papillomavirus (HPV) isolated by manual extraction method (AmpliLute) and automated MagNA pure system were compared and evaluated with cytohistological findings in 253 women. The concordance level between AmpliLute and MagNA was very good 93.3% (κ=0.864,P<.0001). Overall HPVpositivity detected by AmpliLute was 57.3% (30.4% as single and 27% as multiple infections) in contrast to MagNA 54.5% (32% and 23%, resp.). Discrepant results observed in 25 cases: 11 MagNA(−)/AmpliLute(+), 10 of which had positive histology; 5 MagNA(+)/AmpliLute(−) with negative histology; 8 MagNA(+)/AmpliLute(+): in 7 of which AmpliLute detected extra HPV genotypes and 1 MagNA(invalid)/AmpliLute(+) with positive histology. Both methods performed well when compared against cytological (area under curve (AUC) of AmpliLute 0.712 versus 0.672 of MagNA) and histological diagnoses (AUC of AmpliLute 0.935 versus 0.877 of MagNA), with AmpliLute showing a slightly predominance over MagNA. However, higher sensitivities, specificities, and positive/negative predictive values were obtained by AmpliLute.

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