scholarly journals Current Methods for Fluorescence-Based Universal Sequence-Dependent Detection of Nucleic Acids in Homogenous Assays and Clinical Applications

2013 ◽  
Vol 59 (11) ◽  
pp. 1567-1582 ◽  
Author(s):  
Bernd Faltin ◽  
Roland Zengerle ◽  
Felix von Stetten
Keyword(s):  
Critical Evaluation ◽  
Cost Effective ◽  
Clinical Applications ◽  
Clinical Diagnostics ◽  
Detection Methods ◽  
Target Sequence ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Sequence Dependent ◽  
Universal Detection

BACKGROUND Specific and sensitive nucleic acid (NA) testing in research and clinical diagnostics is usually performed by use of labeled oligonucleotide probes. However, the use of target-specific fluorogenic probes increases the cost of analysis. Therefore, universal sequence-dependent (USD) NA detection methods have been developed to facilitate cost-effective target detection using standardized reagents. CONTENT We provide a comprehensive review of the current methods for fluorescence-based USD NA detection. Initially, we focus on the emergence of these methods as a means to overcome the shortcomings of common NA detection methods, such as hydrolysis probes and molecular beacons. Thereafter, we provide a critical evaluation of the individual detection methods. These methods include (a) target amplification with bipartite primers introducing a universal detection tag to the amplicon (UniPrimer PCR, universal fluorescence energy transfer probe PCR, attached universal duplex probe PCR, and universal strand displacement amplification) or combined with bipartite probes comprising a universal detection region (mediator probe PCR, universal strand displacement amplification, universal quenching probe PCR) and (b) amplification-independent assays employing either a universal variant of the invader assay or universal NA hybridization sensors. We discuss differences between the methods and review clinical applications. SUMMARY The current methods for USD NA testing are cost-effective and flexible and have concordant analytical performance in comparison with common probe-based techniques. They can detect any target sequence by the simple use of a label-free, low-cost primer or probe combined with a universal fluorogenic reporter. The methods differ in the number of target specificities, capability of multiplexing, and incubation requirements (isothermal/thermocycling). Extensive clinical applications comprise detection of single-nucleotide polymorphisms, study of gene expression, in situ PCR, and quantification of pathogen load.

scholarly journals Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

1996 ◽  
Vol 42 (10) ◽  
pp. 1604-1608 ◽  
Author(s):  
G T Walker ◽  
C P Linn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Restriction Enzyme ◽  
Fluorescence Polarization ◽  
Target Sequence ◽  
Strand Displacement ◽  
Insertion Element ◽  
Strand Displacement Amplification ◽  
Simple Protocol ◽  
Polymerase Binding

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.

scholarly journals Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽  
2021 ◽  
Vol 21 (12) ◽  
pp. 3985
Author(s):  
Nan Wan ◽  
Yu Jiang ◽  
Jiamei Huang ◽  
Rania Oueslati ◽  
Shigetoshi Eda ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Detection Methods ◽  
Capacitive Sensing ◽  
Application Potential ◽  
Mirna Detection ◽  
Mirna 25 ◽  
Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

scholarly journals Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

Sensors ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 602
Author(s):  
Sandra Leonardo ◽  
Anna Toldrà ◽  
Mònica Campàs
Keyword(s):  
Food Safety ◽  
Environmental Monitoring ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
In Situ Monitoring ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Bacterial Detection ◽  
Efficient Detection

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS): basics and clinical applications

2020 ◽  
Vol 58 (6) ◽  
pp. 883-896 ◽  
Author(s):  
Muhammad Zubair Israr ◽  
Dennis Bernieh ◽  
Andrea Salzano ◽  
Shabana Cassambai ◽  
Yoshiyuki Yazaki ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Maldi Mass Spectrometry ◽  
Clinical Applications ◽  
Clinical Diagnostics ◽  
Maldi Ms ◽  
Therapeutic Drug ◽  
The Impact ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

AbstractBackgroundMatrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS) has been used for more than 30 years. Compared with other analytical techniques, it offers ease of use, high throughput, robustness, cost-effectiveness, rapid analysis and sensitivity. As advantages, current clinical techniques (e.g. immunoassays) are unable to directly measure the biomarker; rather, they measure secondary signals. MALDI-MS has been extensively researched for clinical applications, and it is set for a breakthrough as a routine tool for clinical diagnostics.ContentThis review reports on the principles of MALDI-MS and discusses current clinical applications and the future clinical prospects for MALDI-MS. Furthermore, the review assesses the limitations currently experienced in clinical assays, the advantages and the impact of MALDI-MS to transform clinical laboratories.SummaryMALDI-MS is widely used in clinical microbiology for the screening of microbial isolates; however, there is scope to apply MALDI-MS in the diagnosis, prognosis, therapeutic drug monitoring and biopsy imaging in many diseases.OutlookThere is considerable potential for MALDI-MS in clinic as a tool for screening, profiling and imaging because of its high sensitivity and specificity over alternative techniques.

Ultra-sensitive MicroRNA-21 detection based on multiple cascaded strand displacement amplification and CRISPR/Cpf1 (MC-SDA/CRISPR/Cpf1)

2021 ◽  
Author(s):  
Xiaolong Chen ◽  
Yuanyi Deng ◽  
Gaihua Cao ◽  
Yifan Xiong ◽  
Danqun Huo ◽  
...  
Keyword(s):  
Cancer Diagnosis ◽  
Analytical Method ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Diagnosis And Prognosis ◽  
Potential Biomarker ◽  
Cancer Diagnosis And Prognosis

MicroRNA-21 (miR-21) has been considered as a potential biomarker for cancer diagnosis and prognosis due to its highly expressed in tumors. Here, an analytical method which integrates the multiple cascaded...

scholarly journals mRNA-Based Vaccines

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 390
Author(s):  
Frank Kowalzik ◽  
Daniel Schreiner ◽  
Christian Jensen ◽  
Daniel Teschner ◽  
Stephan Gehring ◽  
...  
Keyword(s):  
Large Scale ◽  
New Technologies ◽  
Vaccine Development ◽  
Effective Means ◽  
Cost Effective ◽  
Clinical Applications ◽  
Cell Mediated Immunity ◽  
Healthy Individuals ◽  
Emerging Pathogens ◽  
Cell Genome

Increases in the world’s population and population density promote the spread of emerging pathogens. Vaccines are the most cost-effective means of preventing this spread. Traditional methods used to identify and produce new vaccines are not adequate, in most instances, to ensure global protection. New technologies are urgently needed to expedite large scale vaccine development. mRNA-based vaccines promise to meet this need. mRNA-based vaccines exhibit a number of potential advantages relative to conventional vaccines, namely they (1) involve neither infectious elements nor a risk of stable integration into the host cell genome; (2) generate humoral and cell-mediated immunity; (3) are well-tolerated by healthy individuals; and (4) are less expensive and produced more rapidly by processes that are readily standardized and scaled-up, improving responsiveness to large emerging outbreaks. Multiple mRNA vaccine platforms have demonstrated efficacy in preventing infectious diseases and treating several types of cancers in humans as well as animal models. This review describes the factors that contribute to maximizing the production of effective mRNA vaccine transcripts and delivery systems, and the clinical applications are discussed in detail.

scholarly journals Microsphere-Based Microfluidic Device for Plasma Separation and Potential Biochemistry Analysis Applications

Micromachines ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 487
Author(s):  
Hongyan Xu ◽  
Zhangying Wu ◽  
Jinan Deng ◽  
Jun Qiu ◽  
Ning Hu ◽  
...  
Keyword(s):  
Blood Sample ◽  
Microfluidic Device ◽  
Biochemical Analysis ◽  
Cost Effective ◽  
Clinical Diagnostics ◽  
Correction Factors ◽  
Separation Device ◽  
Blood Samples ◽  
Plasma Separation ◽  
Blood Biochemical

The development of a simple, portable, and cost-effective plasma separation platform for blood biochemical analysis is of great interest in clinical diagnostics. We represent a plasma separation microfluidic device using microspheres with different sizes as the separation barrier. This plasma separation device, with 18 capillary microchannels, can extract about 3 μL of plasma from a 50 μL blood sample in about 55 min. The effects of evaporation and the microsphere barrier on the plasma biochemical analysis results were studied. Correction factors were applied to compensate for these two effects. The feasibility of the device in plasma biochemical analysis was validated with clinical blood samples.

scholarly journals Food Allergen Detection Methods: A Coordinated Approach

2004 ◽  
Vol 87 (6) ◽  
pp. 1383-1390 ◽  
Author(s):  
Philip R Goodwin
Keyword(s):  
Hazard Analysis ◽  
Adult Population ◽  
Control Point ◽  
Cost Effective ◽  
Test Methods ◽  
Detection Methods ◽  
Critical Control Point ◽  
Life Threatening ◽  
Monitoring And Surveillance ◽  
Analytical Approaches

Abstract The levels (1–2%) and increasing severity of allergic responses to food in the adult population are well documented, as is the phenomenon of even higher (3–8%) and apparently increasing incidence in children, albeit that susceptibility decreases with age. Problematic foods include peanut, milk, eggs, tree nuts, and sesame, but the list is growing as awareness continues to rise. The amounts of such foods that can cause allergic reactions is difficult to gauge; however, the general consensus is that ingestion of low parts per million is sufficient to cause severe reactions in badly affected individuals. Symptoms can rapidly—within minutes—progress from minor discomfort to severe, even life-threatening anaphylactic shock in those worst affected. Given the combination of high incidence of atopy, potential severity of response, and apparently widespread instances of “hidden” allergens in the food supply, it is not surprising that this issue is increasingly subject to legislative and regulatory scrutiny. In order to assist in the control of allergen levels in foods to acceptable levels, analysts require a combination of test methods, each designed to produce accurate, timely, and cost-effective analytical information. Such information contributes significantly to Hazard Analysis Critical Control Point programs to determine food manufacturers’ risk and improves the accuracy of monitoring and surveillance by food industry, commercial, and enforcement laboratories. Analysis thereby facilitates improvements in compliance with labeling laws with concomitant reductions in risks to atopic consumers. This article describes a combination of analytical approaches to fulfill the various needs of these 3 analytical communities.

Sign in / Sign up

Export Citation Format

Share Document