scholarly journals DEVELOPMENT OF DNA BIOSENSOR BASED ON SILVER NANOPARTICLES UV-Vis ABSORPTION SPECTRA FOR Escherichia coli DETECTION

2015 ◽  
Vol 2 (1) ◽  
pp. 382 ◽  
Author(s):  
Ruth Chrisnasari ◽  
Antonius Loren Wijaya ◽  
Maria Goretti Marianti Purwanto
Keyword(s):  
Escherichia Coli ◽  
Silver Nanoparticles ◽  
Absorption Spectra ◽  
Magnetic Beads ◽  
Dna Isolation ◽  
Dna Biosensor ◽  
Isolation Method ◽  
Isolation Methods ◽  
Spectra Properties ◽  
Magnetic Field Condition

<p>In this research we reported the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrated their use along with magnetic beads as biosensor for Escherichia coli detection under magnetic field condition. Oligonucleotide DNA probes were conjugated on silver nanoparticles using alkanethiols linker. Two kinds of alkanethiols linker, 11-mercaptoundodecanoic acid (11-MUDA) and 16-mercaptophexadecanoic acid (16-MHDA) were compared to get the best probe conjugation yield and OSN UV-Vis absorption spectra properties. Three different methods of Escherichia coli DNA isolation i.e. Chen and Kuo (1993), Phenol Chloroform Isoamylalcohol (PCI) extraction and boiling lysis were also compared to explore the performance of the biosensor towards the DNA target purity. Detection process through hybridization between the DNA probe and the target was carried out at 55oC for 1 hour incubation time. The results showed that 16-MHDA gave higher conjugation yield and higher OSN UV-Vis absorption spectra than 11-MUDA. The biosensor was able to detect the presence of the DNA target which was isolated from the three isolation methods. The best detection signal was achieved by Chen and Kuo isolation method in which it could detect the presence of the DNA target up to 1.3 ng/µL.</p><p><br /><strong>Keywords</strong>: DNA biosensor, Silver Nanoparticles, Escherichia coli</p>

scholarly journals Biased nicking of mitochondrial DNA during extraction can be avoided by choice of isolation method

2021 ◽  
Author(s):  
Bruno Marçal Repolês ◽  
Choco Michael Gorospe ◽  
Phong Tran ◽  
Anna Karin Nilsson ◽  
Paulina H. Wanrooij
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Dna ◽  
Dna Isolation ◽  
Isolation Procedure ◽  
Isolation Method ◽  
Strand Breaks ◽  
Careful Choice ◽  
Isolation Methods ◽  
Solid Tissues ◽  
Long Range Pcr

AbstractThe integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR. We show that a commonly-used DNA isolation procedure preferentially introduces strand breaks into the mtDNA extracted from the skeletal muscle of aged mice, while mtDNA from adult animals is less affected. We present a comparison of mtDNA isolation methods and identify one that avoids this biased loss of muscle mtDNA integrity. Our results highlight the importance of a careful choice of mtDNA isolation method and serve as a resource to researchers planning analysis of mtDNA isolated from solid tissues.

scholarly journals Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces

Materials ◽  
2020 ◽  
Vol 13 (22) ◽  
pp. 5112
Author(s):  
Marketa Husakova ◽  
Petr Kralik ◽  
Vladimir Babak ◽  
Iva Slana
Keyword(s):  
Magnetic Separation ◽  
Mycobacterium Avium ◽  
Magnetic Beads ◽  
Dna Isolation ◽  
Mycobacterium Avium Subsp ◽  
Effective Control ◽  
Silica Column ◽  
Faecal Samples ◽  
Isolation Methods ◽  
Mycobacterium Avium Subsp Paratuberculosis

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

scholarly journals Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research

2019 ◽  
Author(s):  
Kari Oline Bøifot ◽  
Jostein Gohli ◽  
Line Victoria Moen ◽  
Marius Dybwad
Keyword(s):  
Microbial Community ◽  
Real World ◽  
Dna Isolation ◽  
New Method ◽  
Metagenomic Sequencing ◽  
Isolation Method ◽  
Air Samples ◽  
Shotgun Metagenomic Sequencing ◽  
Isolation Methods ◽  
Microbiome Research

ABSTRACTBackgroundAerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations.ResultsHere, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures unbiased microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world subway air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway air samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity and gave better taxonomic coverage. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway air samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract.ConclusionsBy demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

scholarly journals Genetic of Salak Pondoh, Gading Varieties and Its Hybrids Based on RAPD Markers

2021 ◽  
Vol 1 (1) ◽  
pp. 5
Author(s):  
Nandariyah Nandariyah ◽  
Parjanto Parjanto ◽  
Pinaka Pinasti Ratu
Keyword(s):  
Molecular Marker ◽  
Rapd Markers ◽  
Dna Isolation ◽  
Dna Amplification ◽  
Isolation Method ◽  
Isolation Methods

<p>A molecular marker of parent and offspring is used to find fast and accurate markers influenced by DNA isolation and amplification. This research aims to find the most suitable DNA isolation and DNA  amplification methods. This study used four DNA isolation methods; namely IM01, IM02, IM03, and IM04. DNA amplification used ten protocols (AP01, AP02, AP03, AP04, AP05, AP06, AP07, AP08, AP09, and AP010). The results of the research showed that the most suitable DNA isolation method for salak was  IM0, and the most suitable DNA amplification for salak was AP04 that produces the highest value of DNA bands.</p><p><strong> </strong></p><p>Keywords: DNA isolation; DNA amplification; hybrids.</p>

Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field

2011 ◽  
Vol 4 (3) ◽  
pp. 1491-1496
Author(s):  
Umadevi M ◽  
Rani T ◽  
Balakrishnan T ◽  
Ramanibai R
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Silver Nanoparticles ◽  
Reduction Method ◽  
Therapeutic Potential ◽  
Chemical Reduction ◽  
Ultrasonic Field ◽  
Great Promise ◽  
Chemical Reduction Method ◽  
E Coli

Nanotechnology has great promise for improving the therapeutic potential of medicinal molecules and related agents. In this study, silver nanoparticles of different sizes were synthesized in an ultrasonic field using the chemical reduction method with sodium borohydride as a reducing agent. The size effect of silver nanoparticles on antimicrobial activity were tested against the microorganisms Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934). The results shows that B. subtilis, and E. coli were more sensitive to silver nanoparticles and its size, indicating the superior antimicrobial efficacy of silver nanoparticles. 

scholarly journals Transcriptome analysis of Escherichia coli K1 after therapy with hesperidin conjugated with silver nanoparticles

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Abdulkader Masri ◽  
Naveed Ahmed Khan ◽  
Muhammad Zarul Hanifah Md Zoqratt ◽  
Qasim Ayub ◽  
Ayaz Anwar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Silver Nanoparticles ◽  
Minimum Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Neonatal Meningitis ◽  
E Coli ◽  
Drug Candidates ◽  
Metabolic Genes ◽  
Genetic Mechanisms ◽  
Cell Stress Response

Abstract Backgrounds Escherichia coli K1 causes neonatal meningitis. Transcriptome studies are indispensable to comprehend the pathology and biology of these bacteria. Recently, we showed that nanoparticles loaded with Hesperidin are potential novel antibacterial agents against E. coli K1. Here, bacteria were treated with and without Hesperidin conjugated with silver nanoparticles, and silver alone, and 50% minimum inhibitory concentration was determined. Differential gene expression analysis using RNA-seq, was performed using Degust software and a set of genes involved in cell stress response and metabolism were selected for the study. Results 50% minimum inhibitory concentration with silver-conjugated Hesperidin was achieved with 0.5 μg/ml of Hesperidin conjugated with silver nanoparticles at 1 h. Differential genetic analysis revealed the expression of 122 genes (≥ 2-log FC, P< 0.01) in both E. coli K1 treated with Hesperidin conjugated silver nanoparticles and E. coli K1 treated with silver alone, compared to untreated E. coli K1. Of note, the expression levels of cation efflux genes (cusA and copA) and translocation of ions, across the membrane genes (rsxB) were found to increase 2.6, 3.1, and 3.3- log FC, respectively. Significant regulation was observed for metabolic genes and several genes involved in the coordination of flagella. Conclusions The antibacterial mechanism of nanoparticles maybe due to disruption of the cell membrane, oxidative stress, and metabolism in E. coli K1. Further studies will lead to a better understanding of the genetic mechanisms underlying treatment with nanoparticles and identification of much needed novel antimicrobial drug candidates.

A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria)

1992 ◽  
Vol 38 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Ken F. Jarrell ◽  
David Faguy ◽  
Anne M. Hebert ◽  
Martin L. Kalmokoff
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Thermal Denaturation ◽  
Dna Isolation ◽  
Methanogenic Archaea ◽  
Restriction Enzymes ◽  
Mole Percent ◽  
High Molecular Weight Dna ◽  
Isolation Methods ◽  
General Method

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol–chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell. Key words: methanogens, archaebacteria, archaea, DNA isolation.

scholarly journals Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0143889 ◽  
Author(s):  
Alexander Tolios ◽  
Daniel Teupser ◽  
Lesca M. Holdt
Keyword(s):  
Telomere Length ◽  
Whole Blood ◽  
Dna Isolation ◽  
Isolation Methods ◽  
Preanalytical Conditions
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