Advances in Nucleic Acid Detection and Quantification

2009 ◽  
Vol 37 (2) ◽  
pp. e1-e4 ◽  
Author(s):  
Ian C. Kavanagh ◽  
Simon C. Baker
Keyword(s):  
Nucleic Acid ◽  
Molecular Biology ◽  
Single Molecule ◽  
Single Cells ◽  
Genetic Research ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
British Library ◽  
Introductory Session ◽  
Detection And Quantification

The last decade has seen many changes in molecular biology at the bench, as we have moved away from a primary goal of cataloguing genes and mRNA towards techniques that detect and quantify nucleic acid molecules even within single cells. With the invention of the polymerase chain reaction (PCR), a nucleic acid sequence could now be amplified to generate a large number of identical copies, and this launched a new era in genetic research. PCR has developed in parallel to fluorescent hybridization probing to provide low-, medium- and high-throughput detection methods. However, PCR and hybridization detection have significant drawbacks as long-term solutions for routine research and diagnostics assays. Therefore many novel methods are being developed independently, but as yet no one technique has emerged as a clear replacement for PCR, microarrays or even sequencing. In order to examine the technological horizon in this area, around 90 delegates assembled at Hinxton Hall, Cambridge, U.K. on 28 and 29 October 2008 for a Biochemical Society/Wellcome Trust Focused Meeting sponsored by Thermo Fisher Scientific and the British Library. The title of the meeting was ‘Advances in Nucleic Acid Detection and Quantification’, and the primary aim was to bring together scientists from different disciplines who nevertheless are trying to develop reliable methods for the quantification or detection of RNA and DNA molecules. This meant that physical and organic chemists, microbial ecologists and clinicians appeared alongside molecular biologists. An introductory session on general nucleic acid detection technologies was initiated with a fascinating insight into single-molecule, singlecell hybridization from Professor Sir Edwin Southern. This served as an ideal base for sessions on single-cell molecular biology and an examination of current applications of emerging technology. This issue of Biochemical Society Transactions contains some of the papers prepared by speakers at the meeting, and highlights not only how PCR and microarrays are already being replaced, but also which methods are likely to replace them.

scholarly journals Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243319
Author(s):  
Takeshi Hanami ◽  
Tetsuya Tanabe ◽  
Takuya Hanashi ◽  
Mitsushiro Yamaguchi ◽  
Hidetaka Nakata ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Detection System ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Digital Measurement ◽  
Excitation Light ◽  
Target Nucleic Acid ◽  
Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

scholarly journals Development and detection of genetically modified crops

2020 ◽  
Vol 145 ◽  
pp. 01013
Author(s):  
Zhao Yu-jia ◽  
Fan Pei-lei ◽  
Liang Liang ◽  
Liu Yin-yin ◽  
Zhao Hai-bo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genetically Modified ◽  
Genetically Modified Crops ◽  
Detection Methods ◽  
Social Benefits ◽  
Nucleic Acid Detection ◽  
Genetically Modified Crop ◽  
The World ◽  
The Difference

Genetically modified crops (GMCs) have been known for the excellent qualities. The commercializing of GMCs has taken great economic and social benefits. However, the bio-security of GMCs was still an issue. To solve this problem, countries around the world were constantly strengthening regulations on planting, processing and detecting of GMCs. This paper reviewed the development of commercialization and detection of GMCs. The difference between protein and nucleic acid detection methods of genetically modified crop was further discussed. This paper will provide new insights for the application of genetically modified crops.

Automated Single Molecule Nucleic Acid Detection with a Waveguide Chip

CLEO: 2014 ◽  
2014 ◽  
Author(s):  
J.W. Parks ◽  
L. Zempoaltecatl ◽  
R. A. Mathies ◽  
A.R. Hawkins ◽  
H. Schmidt
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Nucleic Acid Detection

scholarly journals Evaluation of seven different rapid methods for nucleic acid detection of SARS-COV-2 virus

2021 ◽  
Author(s):  
Sally Mahmoud ◽  
Esra Ibrahim ◽  
Subhashini Ganesan ◽  
Bhagyashree Thakre ◽  
Juliet Teddy ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Gold Standard ◽  
Large Scale ◽  
Virus Detection ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Test Accuracy ◽  
Rt Pcr ◽  
Rapid Methods ◽  
Kappa Value

Background In the current COVID-19 pandemic there is mass screening of SARS-CoV-2 happening round the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite identification of cases and to facilitate early isolation and control spread. Hence this study evaluates seven different rapid nucleic acid detection assays that are commercially available for SARS- CoV- 2 virus detection. Methods Nasopharyngeal samples were collected from 4859 participants and were tested for SARS-CoV-2 virus by the gold standard RT-PCR method along with one of these seven rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. Results AQ-TOP had the highest sensitivity (98%) and strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Conclusion Genechecker system, Abbott ID NOW and Cobas Liat, were found to have best performance and agreement when compared to the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large scale screening of COVID-19.

scholarly journals Assessment of the Diagnostic Ability of Four Detection Methods Using Three Sample Types of COVID-19 Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Fei Yu ◽  
Guoliang Xie ◽  
Shufa Zheng ◽  
Dongsheng Han ◽  
Jiaqi Bao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Sample Type ◽  
Rt Pcr ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Oropharyngeal Swab ◽  
And Control

BackgroundViral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. However, unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic.MethodsWe compared four nucleic acid detection methods [two kinds of reverse transcription polymerase chain reactions (RT-PCR A: ORF1ab and N testing; RT-PCRB: only ORF1ab testing), reverse transcription recombinase aided amplification (RT-RAA) and droplet digital RT-PCR (dd-RT-PCR)] using 404 samples of 72 hospitalized COVID-19 patients, including oropharyngeal swab (OPS), nasopharyngeal swabs (NPS) and saliva after deep cough, to evaluate the best sample type and method for SARS-CoV-2 detection.ResultsAmong the four methods, dd-RT-PCR exhibited the highest positivity rate (93.0%), followed by RT-PCR B (91.2%) and RT-RAA (91.2%), while the positivity rate of RT-PCR A was only 71.9%. The viral load in OPS [24.90 copies/test (IQR 15.58-129.85)] was significantly lower than that in saliva [292.30 copies/test (IQR 20.20-8628.55)] and NPS [274.40 copies/test (IQR 33.10-2836.45)]. In addition, if OPS samples were tested alone by RT-PCR A, only 21.4% of the COVID-19 patients would be considered positive. The accuracy of all methods reached nearly 100% when saliva and NPS samples from the same patient were tested simultaneously.ConclusionsSARS-CoV-2 nucleic acid detection methods should be fully evaluated before use. High-positivity rate methods such as RT-RAA and dd-RT-PCR should be considered when possible. Furthermore, saliva after deep cough and NPS can greatly improve the accuracy of the diagnosis, and testing OPS alone is not recommended.

scholarly journals Analytical Ancestry: “Firsts” in Fluorescent Labeling of Nucleosides, Nucleotides, and Nucleic Acids

2009 ◽  
Vol 55 (4) ◽  
pp. 670-683 ◽  
Author(s):  
Larry J Kricka ◽  
Paolo Fortina
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Fluorescent Dyes ◽  
Fluorescent Labeling ◽  
Covalent Attachment ◽  
Detection Methods ◽  
Fluorescent Label ◽  
Nucleic Acid Detection ◽  
Fluorescent Labels ◽  
Fluorescent Properties

Abstract Background: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications. Content: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals. Summary: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Virus Detection: A Review of the Current and Emerging Molecular and Immunological Methods

2021 ◽  
Vol 8 ◽  
Author(s):  
A. Cassedy ◽  
A. Parle-McDermott ◽  
R. O’Kennedy
Keyword(s):  
Nucleic Acid ◽  
Virus Detection ◽  
High Sensitivity ◽  
High Volume ◽  
Detection Methods ◽  
Immunological Methods ◽  
Nucleic Acid Detection ◽  
Viral Genomes ◽  
Diagnostic Measures ◽  
Point Of Use

Viruses are ubiquitous in the environment. While many impart no deleterious effects on their hosts, several are major pathogens. This risk of pathogenicity, alongside the fact that many viruses can rapidly mutate highlights the need for suitable, rapid diagnostic measures. This review provides a critical analysis of widely used methods and examines their advantages and limitations. Currently, nucleic-acid detection and immunoassay methods are among the most popular means for quickly identifying viral infection directly from source. Nucleic acid-based detection generally offers high sensitivity, but can be time-consuming, costly, and require trained staff. The use of isothermal-based amplification systems for detection could aid in the reduction of results turnaround and equipment-associated costs, making them appealing for point-of-use applications, or when high volume/fast turnaround testing is required. Alternatively, immunoassays offer robustness and reduced costs. Furthermore, some immunoassay formats, such as those using lateral-flow technology, can generate results very rapidly. However, immunoassays typically cannot achieve comparable sensitivity to nucleic acid-based detection methods. Alongside these methods, the application of next-generation sequencing can provide highly specific results. In addition, the ability to sequence large numbers of viral genomes would provide researchers with enhanced information and assist in tracing infections.

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