Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Label-free detection of microRNA based on coupling multiple isothermal amplification techniques.

ABSTRACT: MicroRNA (miRNA) was a promising class of cancer biomarkers. Here we developed a label-free method for sensitive measurement of let-7d miRNA based on multiple amplification techniques. The primer will bind to the duplex strand DNA that was formed by stem-loop template and target let-7d to initiate strand displacement amplification (SDA) in tandem. The released single strand DNA will be a primer to bind the circular template to initiate rolling circle amplification (RCA). The products based on multiple amplifications will be detected by a standard fluorimeter with N-methyl mesoporphyrin IX (NMM) as the fluorescent indicator. The proposed method exhibited excellent selectivity and high sensitivity with a detection limit of as low as 1.5?×?10^-13?M. Moreover, this methodology was used for the determination of biomolecules in real serum samples with satisfying results.

SUBMITTER: Zheng X

PROVIDER: S-EPMC5078768 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Label-free detection of microRNA based on coupling multiple isothermal amplification techniques.

Zheng Xiangjiang X Niu Li L Wei Di D Li Xuemei X Zhang Shusheng S

Scientific reports 20161025

MicroRNA (miRNA) was a promising class of cancer biomarkers. Here we developed a label-free method for sensitive measurement of let-7d miRNA based on multiple amplification techniques. The primer will bind to the duplex strand DNA that was formed by stem-loop template and target let-7d to initiate strand displacement amplification (SDA) in tandem. The released single strand DNA will be a primer to bind the circular template to initiate rolling circle amplification (RCA). The products based on mu ...[more]

PMID: 27777399

Similar Datasets

Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection.

Project description:We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer.

| S-EPMC5046170 | biostudies-literature

An integrated E-Tube cap for sample preparation, isothermal amplification and label-free electrochemical detection of DNA.

Project description:A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6-9 with a slope of -31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

| S-EPMC8572321 | biostudies-literature

An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

Project description:Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

| S-EPMC7127409 | biostudies-literature

Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures.

Project description:We report on a single-tube biosensor for real-time detection of bacterial pathogens with multiplex capabilities. The biosensor consists of two DNA probes, which bind to the complementary fragment of a bacterial RNA to form a three-way junction (3WJ) nucleic acid structure. One of the probes encodes a fluorescent light-up RNA aptamer under T7 promoter. It allows for generation of multiple aptamer copies due to elongation and transcription of the 3WJ structure in the presence of the complementary target. The aptamer coordinates and thereby enhances fluorescence of a cognate fluorogenic dye, allowing for fluorescent detection of the RNA target. Multiple aptamer copies can be produced from a single target-dependent 3WJ structure allowing for amplification and visual observation of the signal. The limit of detection depended on the assay time and was found to be 1.7 nM or 0.6 nM for 30-min or 60-min assay, respectively, when N-methylmesoporphyrin IX (NMM) was used as a fluorescent indicator. The sensor is excellent in analyzing folded RNA targets and differentiating between closely related sequences due to the multicomponent character of the target-interrogating probe. Response to unamplified samples of total bacterial RNA from Mycobacterium tuberculosis complex or Escherichia coli was observed with excellent selectivity within 30 min under isothermal conditions at 50°C in a one-tube one-step assay. Several bacterial species can be detected in multiplex by utilizing biosensors with the template strands encoding different light-up aptamers. The isothermal one-tube-one-step format of the assay and the possibility to monitor the signal visually makes it amenable to use in a point-of-care scenario.

| S-EPMC9475119 | biostudies-literature

Reliable detection of Burkholderia pseudomallei using multiple cross displacement amplification label-based biosensor.

Project description:BackgroundBurkholderia pseudomallei (B. pseudomallei), as a highly pathogenic organism, causes melioidosis, which is a disease of public health importance in many tropical developing countries. Here, we present and validate a novel detection technique, termed multiple cross displacement amplification combined with nanoparticles-based lateral flow biosensor (MCDA-NB), for identifying B. pseudomallei and diagnosing melioidosis.ResultsB. pseudomallei-MCDA targets the TTS1 (Type III secretion system gene cluster 1) to specifically design ten MCDA primers. The nanoparticles-based biosensor (NB) can be combined with B. pseudomallei-MCDA for visually, objective, simply and rapidly reporting reaction results. The optimal amplification conditions of B. pseudomallei-MCDA were 66 °C for 30 min. Assay's sensitivity was 100 fg of genomic DNA in the pure cultures, and the analytical specificity was 100% by the examination of 257 strains, including 228 B. pseudomallei and 29 non-B. pseudomallei. As a result, the whole detection procedure was completed within 50 min, including 15 min for genomic DNA preparation, 30 min for l MCDA reaction, and 2 min for the interpretation of the results visually by biosensor.ConclusionsB. pseudomallei-MCDA assay is a rapid, sensitive and specific method for the detection of B. pseudomallei, and can be used as a potential tool for melioidosis diagnose in basic, field and clinical laboratories.

| S-EPMC8908694 | biostudies-literature

A G-quadruplex DNA-based, label-free and ultrasensitive strategy for microRNA detection.

Project description:MicroRNAs (miRNAs) have been considered to be potent biomarkers for early disease diagnosis and for cancer therapy. The rapid and selective detection of miRNAs without reverse transcription and labelling is highly desired. Herein, we report a simple and label-free miRNA detection method that is based on the Duplex-Specific Nuclease (DSN)-Assisted simple target miRNA recycling procedure. The interaction of the G-quadruplex DNA structure with N-methyl mesoporphyrin IX (NMM) led to a label-free signal output. Under the optimised conditions, this method allowed for simple, rapid, and sequence-specific detection of miR-141 over a dynamic range from 1 fM to 100 nM with a linear range from 1 pM to 100 nM. Moreover, our method offered an excellent capacity to discriminate between miRNA family members with just one mismatched nucleotide. This simple and label-free strategy holds great potential in applications in biomedical research and in early clinical diagnostics.

| S-EPMC4261168 | biostudies-literature

Fluorescent on-site detection of multiple pathogens using smartphone-based portable device with paper-based isothermal amplification chip.

Project description:The development of cost-effective, portable, and ease-of-use sensing system for on-site genetic diagnostics is highly desirable for pathogen screening and infectious disease diagnosis. This study develops (1) a paper-based biochip which is able to integrate the loop-mediated isothermal amplification (LAMP) protocols for simultaneous detection of Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus, and (2) a stand-alone smartphone-based portable device which can control exactly 65 °C for isothermal amplification as well as collect and analyze the thus generated fluorescence signals. The reported sensing system has been successfully demonstrated for foodborne pathogen detection with a limit of detection of 2.8 × 10-5 ng μL-1. Spiked milk samples with concentration as low as 10 CFU mL-1 were successfully determined within 4 h, demonstrating the practicality of the reported sensing system in the fields. The reported sensing system featuring simplicity and reliability is ideally suited for genetic diagnostics in low resource settings.

| S-EPMC9378262 | biostudies-literature

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

Project description:We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

| S-EPMC4648395 | biostudies-other

Facile and Label-Free Electrochemical Biosensors for MicroRNA Detection Based on DNA Origami Nanostructures.

Project description:MicroRNAs (miRNAs) have emerged as the promising molecular biomarkers for early diagnosis and enhanced understanding of the molecular pathogenesis of cancers as well as certain diseases. Here, a facile, label-free, and amplification-free electrochemical biosensor was developed to detect miRNA by using DNA origami nanostructure-supported DNA probes, with methylene blue (MB) serving as the hybridization redox indicator, for the first time. Specifically, the use of cross-shaped DNA origami nanostructures containing multiple single-stranded DNA probes at preselected locations on each DNA nanostructure could increase the accessibility and the recognition efficiency of the probes (due to the rational controlled density of DNA probes). The successful immobilization of DNA origami probes and their hybridization with targeted miRNA-21 molecules was confirmed by electrochemical impedance spectroscopy and cyclic voltammetry methods. A differential pulse voltammetry technique was employed to record the oxidation peak current of MB before and after target hybridization. The linear detection range of this biosensor was from 0.1 pM to 10.0 nM, with a lower detection limit of 79.8 fM. The selectivity of the miRNA biosensor was also studied by observing the discrimination ability of single-base mismatched sequences. Because of the larger surface area and unprecedented customizability of DNA origami nanostructures, this strategy demonstrated great potential for sensitive, selective, and label-free determination of miRNA for translational biomedical research and clinical applications.

| S-EPMC6649092 | biostudies-literature

Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.

Project description:Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~10(9) copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a nonenzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both nonspecific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons.

| S-EPMC3478682 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data