scholarly journals Isothermal digital detection of microRNAs using background-free molecular circuit

2020 ◽  
Vol 6 (4) ◽  
pp. eaay5952 ◽  
Author(s):  
Guillaume Gines ◽  
Roberta Menezes ◽  
Kaori Nara ◽  
Anne-Sophie Kirstetter ◽  
Valerie Taly ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Isothermal Amplification ◽  
Absorption Mechanism ◽  
Molecular Programming ◽  
Bodily Fluids ◽  
Microrna Detection ◽  
Nonspecific Amplification ◽  
Femtomolar Concentration ◽  
Accurate Quantification ◽  
Digital Counting

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

scholarly journals Isothermal digital detection of microRNA using background-free molecular circuit

2019 ◽  
Author(s):  
Guillaume Gines ◽  
Roberta Menezes ◽  
Kaori Nara ◽  
Anne-Sophie Kirstetter ◽  
Valérie Taly ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Isothermal Amplification ◽  
Absorption Mechanism ◽  
Digital Readout ◽  
Molecular Programming ◽  
Bodily Fluids ◽  
Specific Amplification ◽  
Femtomolar Concentration ◽  
Accurate Quantification ◽  
Digital Counting

AbstractMicroRNA, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: by partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

Rapid MicroRNA Detection Using Paper-Based Isothermal Amplification

2021 ◽  
Author(s):  
Jingjing Qian ◽  
Qinming Zhang ◽  
Joyce C. Lai ◽  
Yixuan Wang ◽  
Meng Lu
Keyword(s):  
Isothermal Amplification ◽  
Microrna Detection

Isothermal Amplification for MicroRNA Detection: From the Test Tube to the Cell

2017 ◽  
Vol 50 (4) ◽  
pp. 1059-1068 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Jinghong Li
Keyword(s):  
Test Tube ◽  
Isothermal Amplification ◽  
Microrna Detection

scholarly journals rEXPAR: an isothermal amplification scheme that is robust to autocatalytic parasites

2019 ◽  
Author(s):  
Georg Urtel ◽  
Jean-Christophe Galas ◽  
André Estevez-Torres
Keyword(s):  
Programming Languages ◽  
Dna Polymerase ◽  
Reaction Network ◽  
Isothermal Amplification ◽  
Sequence Design ◽  
Molecular Programming ◽  
Nicking Enzyme

ABSTRACTIn the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR, as well as in related molecular programming languages, such as the PEN DNA toolbox. Here we show that the nicking enzyme Nb.BssSI allows to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network.

Attomolar Ultrasensitive MicroRNA Detection by DNA-Scaffolded Silver-Nanocluster Probe Based on Isothermal Amplification

2012 ◽  
Vol 84 (12) ◽  
pp. 5165-5169 ◽  
Author(s):  
Yu-Qiang Liu ◽  
Min Zhang ◽  
Bin-Cheng Yin ◽  
Bang-Ce Ye
Keyword(s):  
Isothermal Amplification ◽  
Silver Nanocluster ◽  
Microrna Detection

scholarly journals A Novel Isothermal Amplification Method for Detecting Mouse Source Component in Meat

2019 ◽  
Vol 102 (3) ◽  
pp. 872-877 ◽  
Author(s):  
Xinnan Li ◽  
Xueqin Gao ◽  
Yifu Guan
Keyword(s):  
Isothermal Amplification ◽  
New Method ◽  
Simple Method ◽  
New Approach ◽  
Safety Issues ◽  
Amplification Method ◽  
Specificity And Sensitivity ◽  
Great Specificity ◽  
Nonspecific Amplification ◽  
Dna Template

Abstract Background: The problem of adulterated meat has become one of the greatest food safety issues in the world. It is reported that the meat used for adulteration includes fox meat, raccoon meat, mink meat, mouse meat, and so on. Although this kind of meat is edible in some areas, such meat is potentially harmful to human health because it is easy to carry bacteria, viruses, and harmful substances. The harm of mouse meat is most frightening. Therefore, it is urgent to develop a fast, accurate, and simple method to effectively identify mouse meat. Methods: In the present study, a new method of isothermal amplification based on the 16S ribosomal RNA gene of the mitochondrial DNA of the mouse was developed. The method is meant to improve the loop-mediated isothermal amplification (LAMP), separating the forward inner primers and backward inner primers, greatly reducing the nonspecific amplification of the method. Results: We have successfully obtained a set of best primers. The developed system allowed for the detection of 0.5% mouse meat from meat mixture effectively and specifically. The best ratio of the primers (F3: F2: F1: RF) was 1:2:2:8, and the optimum concentration of DNA template was 0.35 ng/μL. Conclusions: The assay has great specificity and sensitivity for detecting mouse meat and could provide specific positive results within 1 h. Highlights: We found a new approach of isothermal amplification to detect mouse source components. The LOD is determined to be 0.5 mg/mg. This new method is easy to perform and is able to provide rapid results in the specific detection of mouse meat sources.

MicroRNA detection at femtomolar concentrations with isothermal amplification and a biological nanopore

Nanoscale ◽  
2017 ◽  
Vol 9 (42) ◽  
pp. 16124-16127 ◽  
Author(s):  
Haolin Zhang ◽  
Moe Hiratani ◽  
Kentaro Nagaoka ◽  
Ryuji Kawano
Keyword(s):  
Isothermal Amplification ◽  
Microrna Detection ◽  
Low Concentrations

This paper describes a method to detect ultra-low concentrations of nucleotides using isothermal amplification and a biological nanopore.

A ligation-based loop-mediated isothermal amplification (ligation-LAMP) strategy for highly selective microRNA detection

2016 ◽  
Vol 52 (86) ◽  
pp. 12721-12724 ◽  
Author(s):  
Wenfang Du ◽  
Mengmei Lv ◽  
Junjie Li ◽  
Ruqin Yu ◽  
Jianhui Jiang
Keyword(s):  
Isothermal Amplification ◽  
Lamp Method ◽  
Selective Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Microrna Detection

A novel ligation-based loop-mediated isothermal amplification (ligation-LAMP) method has been developed for sensitive and selective detection of microRNA.

scholarly journals The role of miRNA in selected tumors development: Potential use in diagnostics

2021 ◽  
Vol 75 ◽  
pp. 1-11
Author(s):  
Patrycja Paciorek ◽  
Mariusz Żuberek ◽  
Agnieszka Grzelak
Keyword(s):  
Gene Expression ◽  
Mirna Expression ◽  
Regulation Of Gene Expression ◽  
Regulatory Function ◽  
Cancer Information ◽  
Bodily Fluids ◽  
Experimental Therapies ◽  
Oncological Diagnostics ◽  
Potential Use

MicroRNAs (miRNAs) are small ribonucleic acid molecules that, although not translated, perform an important regulatory function in eukaryotic cells. Their physiological function is to maintain cell homeostasis. Impaired miRNA expression can cause the development of many diseases including cancer. MiRNA biological activity is based on inhibiting the formation of proteins, including oncogenic and anti-oncogenic proteins. Mutations at the coding sites for such miRNAs can lead to overproduction or reduction of the production of the above-mentioned proteins. The discovery of miRNAs and understanding their role in the cell opened new ways for diagnosing cancer. Therefore, changes in the level of relevant miRNAs in the bloodstream or other bodily fluids can be a diagnostic marker of disease. Oncological diagnostics could be based on examining the patient’s miRNA profile and comparing it with previously developed profiles of miRNAs changes associated with the occurrence of a given type of cancer. Information on changes in miRNA profiles that are key to regulating gene expression associated with tumorigenic processes could contribute to the development of experimental therapies based on restoring the original level of miRNA in cells and thereby restoring normal regulation of gene expression. New methods of silencing and enabling miRNA expression may, in the future, result in effective therapeutic solutions.

scholarly journals Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study

2016 ◽  
Vol 54 (8) ◽  
pp. 1984-1991 ◽  
Author(s):  
Christen M. Gray ◽  
Achilles Katamba ◽  
Pratibha Narang ◽  
Jorge Giraldo ◽  
Carlos Zamudio ◽  
...  
Keyword(s):  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Smear Microscopy ◽  
Eligibility Criteria ◽  
Loop Mediated Isothermal Amplification ◽  
Root Cause ◽  
Operational Characteristics ◽  
Reaction Volumes ◽  
Nonspecific Amplification ◽  
Eiken Chemical

Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary.

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