scholarly journals Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

2019 ◽  
Vol 47 (21) ◽  
pp. e141-e141 ◽  
Author(s):  
Antoine Daunay ◽  
Alex Duval ◽  
Laura G Baudrin ◽  
Olivier Buhard ◽  
Victor Renault ◽  
...  
Keyword(s):  
Low Temperature ◽  
Microsatellite Instability ◽  
Tandem Repeats ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Dna Repeat ◽  
Cost Effective Approach ◽  
Living Organisms ◽  
Artifact Formation

Abstract Microsatellites are polymorphic short tandem repeats of 1–6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as ‘stutter peaks’ caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.

scholarly journals Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

2022 ◽  
Author(s):  
Rebecca Allsopp ◽  
Georgios Alexandrou ◽  
Christofer Toumazou ◽  
Simak Ali ◽  
Charles Coombes ◽  
...  
Keyword(s):  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Loop Formation ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Allele ◽  
Allele Specific ◽  
Specific Reactions ◽  
Circulating Tumour Dna

Abstract Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Role of Microorganisms in Bioremediation

2021 ◽  
Vol 2 (2) ◽  
pp. 140-176
Author(s):  
Komal Aslam ◽  
Hafiza Iqra Saeed ◽  
Jessica Alyas ◽  
Aysha Saeed ◽  
Tanveer Majeed ◽  
...  
Keyword(s):  
Organic Pollutants ◽  
Cost Effective ◽  
Industrial Contaminants ◽  
Optimal Functioning ◽  
Cost Effective Approach ◽  
Different Types ◽  
Living Organisms

Bioremediation involves the use of natural microorganisms for the purpose ofdegrading numerous types of industrial and environmental waste. Microorganismsrequire carbon, nutrients, and energy to live and multiply as all living organisms do. Inorder to obtain energy, such microorganisms break down organic pollutants into simplerorganic compounds like carbon, salts, water, and similar harmless products. Thisapproach of degrading contaminants using microorganisms has proved much beneficialand has been proven to be cost-effective and efficient. There are a lot of naturally occurringmicroorganisms that have been reported essential in the degradation of organic pollutants.Different industries use different types of bioremediation methods. Specificenvironmental conditions may be required for optimal functioning of microbes e.g., pH,temperature, humidity, etc. Bioremediation has been proven as an environment-friendlyand cost-effective approach to deal with industrial contaminants. Descriptive informationof microbes involved in bioremediation has been explained in this review.

scholarly journals Cytotoxicity and Bioremediation of Heavy Metals by Highly Resistant Marine Bacteria

2021 ◽  
pp. 48-72
Author(s):  
Enas N. Danial ◽  
Walaa A Majrashi ◽  
Ahlam O. Bin Afif ◽  
Ebtehal S Alamri ◽  
Entesar M. Alhatimi ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Marine Bacteria ◽  
Environmental Concern ◽  
Cost Effective ◽  
Human Beings ◽  
Toxic Heavy Metals ◽  
Cost Effective Approach ◽  
Good Potential ◽  
Living Organisms ◽  
Detoxification Mechanisms

Environmental pollution of heavy metals is increasingly becoming a problem and has become of great concern due to the adverse effects it is causing around the world. These inorganic pollutants are being discarded in our waters, soils and into the atmosphere due to the rapidly growing agriculture and metal industries, improper waste disposal, fertilizers, and pesticides. Pollution in industrial areas is a serious environmental concern. Wastewater containing biotoxic substances of heavy metals in the ecosystem is one of the most important environmental and health challenges in our society. Hence, there is a growing need for the development of novel, efficient, eco-friendly, and cost-effective approach for the remediation of inorganic metals (Cr, Hg, Cd, and Pb) released into the environment and to safeguard the ecosystem. Mercury (Hg), Chromium (Cr), Cadmium (Cd), and lead (Pb) are known to cause damage to living organisms, including human beings. In this regard, recent advances in microbes-base heavy metal have propelled bioremediation as a prospective alternative to conventional techniques. Heavy metals are nonbiodegradable and could be toxic to microbes. Several microorganisms have evolved to develop detoxification mechanisms to counter the toxic effects of these inorganic metals. Several marine bacteria highly resistant and capable of growing at higher concentrations of Hg, Cr, Cd and Pb and to evaluate their potential to detoxify. Their detoxification efficiency for Hg, Cr, Cd and Pb indicates good potential for application in bioremediation of toxic heavy metals.

scholarly journals A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

2021 ◽  
Vol 9 ◽  
Author(s):  
Xin Zheng ◽  
Shunxiang Gao ◽  
Jihong Wu ◽  
Xiaobo Hu
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Disease Diagnosis ◽  
Serum Samples ◽  
Signal Molecule ◽  
Diagnosis And Prognosis ◽  
Cost Effective Approach ◽  
G Quadruplex ◽  
Diabetic Eye Disease ◽  
Fluorescent Aptasensor

VEGF165, a regulator of angiogenesis, has been widely used as a serum biomarker for a number of human diseases, including cancer, rheumatoid arthritis, bronchial asthma, and diabetic eye disease. The rapid, accurate, and convenient detection of VEGF165 is a crucial step in effective healthcare monitoring, disease diagnosis, and prognosis assessment. In this study, a fluorescent aptasensor based on an assembled G-quadruplex and the signal molecule ThT was developed for VEGF165 detection. First, G-rich DNA fragments were assembled at both ends of the anti-VEGF165 aptamer, and the B-DNA form was converted into a G-quadruplex structure aptamer (G4-Apt). Then, ThT was introduced, and the G-quadruplex significantly enhanced the fluorescence intensity of the bound ThT. When VEGF165 was present, the higher affinity of the aptamer to the target protein allowed the G4-Apt/VEGF165 complex to form and release ThT, which emitted only weak fluorescence in the free state. Therefore, the aptasensor exhibited a good linear detection window from 1.56 to 25 nM VEGF165, with a limit of detection of 0.138 nM. In addition, the aptasensor was applied to detect VEGF165 in clinical serum samples, showing good accuracy, reproducibility, and stability. These results indicate that our developed fluorescent aptasensor can potentially be a reliable, convenient, and cost-effective approach for the sensitive, specific, and rapid detection of the VEGF165 biomarker.

scholarly journals Self-Assembly of Functional Nucleic Acid-Based Colorimetric Competition Assay for the Detection of Immunoglobulin E

Sensors ◽  
2019 ◽  
Vol 19 (10) ◽  
pp. 2224 ◽  
Author(s):  
Xuexia Lin ◽  
Caiyun Yu ◽  
Honggui Lin ◽  
Cui Wang ◽  
Jianlong Su ◽  
...  
Keyword(s):  
Self Assembly ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Colorimetric Assay ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cost Effective Approach ◽  
G Quadruplex ◽  
Functional Nucleic Acids

In this work, we have developed a simple and rapid colorimetric assay for the detection of immunoglobulin E (IgE) using functional nucleic acids (FNAs) and a solid-phase competition enzyme-linked immunosorbent assay (ELISA). The FNAs including aptamer of recombinant IgE, G-quadruplex and its complementary fragments were immobilized on 96-well microplates to achieve recognition and detection of IgE in biological samples. The G-quadruplex DNAzyme catalyzed 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-hemin-H2O2 system was used to improve the sensitivity of colorimetric assay. In the presence of IgE, the hairpin structure and G-quadruplex would be destroyed, resulting in the inactivation of DNAzyme and subsequent reduction of its absorbance. This cost-effective approach detected IgE in the linear range from 5.0 pg/mL to 500 ng/mL, with the limit of detection (LOD) of 2.0 pg/mL, under optimal conditions. Moreover, the developed method was successfully applied to the rapid detection of IgE in human urine, indicating a great potentiality of this approach in clinical diagnosis and other biomedical applications.

scholarly journals Cry Protein Crystal-Immobilized Metallothioneins for Bioremediation of Heavy Metals from Water

Crystals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 287 ◽  
Author(s):  
Qian Sun ◽  
Sze Wan Cheng ◽  
Kelton Cheung ◽  
Marianne M. Lee ◽  
Michael K. Chan
Keyword(s):  
Heavy Metals ◽  
Tandem Repeats ◽  
Binding Capacity ◽  
Cost Effective ◽  
Protein Crystal ◽  
Toxic Heavy Metals ◽  
Cry Protein ◽  
Cost Effective Approach ◽  
New Strategy ◽  
Pcc 7942

Cry proteins have been the subject of intense research due to their ability to form crystals naturally in Bacillus thuringiensis (Bt). In this research we developed a new strategy that allows for the removal of cadmium and chromium from wastewater by using one Cry protein, Cry3Aa, as a framework to immobilize tandem repeats of the cyanobacterial metallothionein SmtA from Synechococcus elongatus (strain PCC 7942). SmtA is a low molecular weight cysteine-rich protein known to bind heavy metals. A series of Cry3Aa-SmtA constructs were produced by the fusion of one, three, or six tandem repeats of SmtA to Cry3Aa. Overexpression of these constructs in Bt resulted in the production of pure Cry3Aa-SmtA fusion crystals that exhibited similar size, crystallinity, and morphology to that of native Cry3Aa protein crystals. All three Cry3Aa-SmtA constructs exhibited efficient binding to cadmium and chromium, with the binding capacity correlated with increasing SmtA copy number. These results suggest the potential use of Cry3Aa-SmtA crystals as a novel biodegradable and cost-effective approach to the removal of toxic heavy metals from the environment.

scholarly journals Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2187
Author(s):  
Paulina Rajko-Nenow ◽  
Emma L. A. Howson ◽  
Duncan Clark ◽  
Natasha Hilton ◽  
Aruna Ambagala ◽  
...  
Keyword(s):  
Real Time ◽  
Rapid Detection ◽  
High Throughput Screening ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Cost Effective ◽  
Virus Genome ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Haemorrhagic Disease

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

Sign in / Sign up

Export Citation Format

Share Document