scholarly journals A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0220091 ◽  
Author(s):  
Florian Katzmeier ◽  
Lukas Aufinger ◽  
Aurore Dupin ◽  
Jorge Quintero ◽  
Matthias Lenz ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Low Cost ◽  
In Vitro Transcription ◽  
Nucleic Acid Detection

scholarly journals A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

2019 ◽  
Author(s):  
Florian Katzmeier ◽  
Lukas Aufinger ◽  
Aurore Dupin ◽  
Jorge Quinteiro ◽  
Matthias Lenz ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Nucleic Acid Detection ◽  
Fluorescence Detector ◽  
Flat Detector ◽  
Low Resource Settings ◽  
Low Resource

AbstractPoint-of-care testing (POCT) in low-resource settings requires tools that can operate independent of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably detected less than 10 nM fluorescein concentrations (with a lower limit of detection of ≈6.8 nM), which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs in the nanomolar range using a Cas13a-based fluorescence assay. Cas13a is a RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device architecture could be a valuable educational tool that integrates hardware, software and biochemistry concepts.

scholarly journals Nucleic acid detection systems for enteroviruses.

1991 ◽  
Vol 4 (2) ◽  
pp. 156-168 ◽  
Author(s):  
H A Rotbart
Keyword(s):  
Nucleic Acid ◽  
Slow Growth ◽  
Acid Synthesis ◽  
In Vitro Transcription ◽  
Nucleic Acid Detection ◽  
Human Pathogens ◽  
Detection Systems ◽  
The Past ◽  
Polymerase Chain

The enteroviruses comprise nearly 70 human pathogens responsible for a wide array of diseases including poliomyelitis, meningitis, myocarditis, and neonatal sepsis. Current diagnostic tests for the enteroviruses are limited in their use by the slow growth, or failure to grow, of certain serotypes in culture, the antigenic diversity among the serotypes, and the low titer of virus in certain clinical specimens. Within the past 6 years, applications of molecular cloning techniques, in vitro transcription vectors, automated nucleic acid synthesis, and the polymerase chain reaction have resulted in significant progress toward nucleic acid-based detection systems for the enteroviruses that take advantage of conserved genomic sequences across many, if not all, serotypes. Similar approaches to the study of enteroviral pathogenesis have already produced dramatic advances in our understanding of how these important viruses cause their diverse clinical spectra.

Duplex-specific nuclease-mediated target recycling amplification for fluorescence detection of microRNA

2019 ◽  
Vol 11 (2) ◽  
pp. 200-204 ◽  
Author(s):  
Lin Tan ◽  
Liu Xu ◽  
Jin-Wen Liu ◽  
Li-Juan Tang ◽  
Hao Tang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Detection ◽  
Low Cost ◽  
Isothermal Amplification ◽  
Nucleic Acid Detection ◽  
Target Recycling ◽  
Recycling Amplification

Isothermal amplification techniques for nucleic acid detection have drawn increasing interest recently due to the simplicity and low-cost of instruments.

scholarly journals A low-cost smart system for electrophoresis-based nucleic acids detection at the visible spectrum

2020 ◽  
Author(s):  
Eduardo Nogueira Cunha ◽  
Maria Fernanda Bezerra de Souza ◽  
Daniel Carlos Ferreira Lanza ◽  
João Paulo Matos Santos Lima
Keyword(s):  
Nucleic Acid ◽  
Point Of Care ◽  
Low Cost ◽  
Visible Spectrum ◽  
Nucleic Acid Detection ◽  
Additional Risk Factor ◽  
Documentation System ◽  
Smart System ◽  
Wide Range ◽  
Image Capturing

ABSTRACTNucleic acid detection by electrophoresis is still a quick and accessible technique for many diagnosis methods, primarily at research laboratories or at the point of care units. Standard protocols detect DNA/RNA molecules through specific bound chemical dyes using a UV-transilluminator or UV-photo documentation system. However, the acquisition costs and availability of these devices, mainly the ones with photography and internet connection capabilities, can be prohibitive, especially in developing countries public health units. Also, ultraviolet radiation is a common additional risk factor to professionals that use electrophoresis-based nucleic acid detection. With that in mind, this work describes the development of a low-cost DNA/RNA detection smart system capable of obtaining qualitative and semi-quantitative data from gel analysis. The proposed device explores the visible light absorption range of commonly used DNA/RNA dyes using readily available parts, and simple manufacturing processes, such as light-emitting diodes (LEDs) and 3D impression. By applying IoT techniques, our system covers a wide range of color spectrum in order to detect bands from various commercially used dyes, using Bluetooth communication and a smartphone for hardware control, image capturing, and sharing. The project also enables process scalability and has low manufacturing and maintenance costs. The use of LEDs at the visible spectrum can achieve very reproducible images, providing a high potential for rapid and point-of-care diagnostics as well as applications in several fields such as healthcare, agriculture, and aquaculture.

scholarly journals Characterization of Argonaute nucleases from mesophilic bacteria Paenibacillus borealis and Brevibacillus laterosporus

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Huarong Dong ◽  
Fei Huang ◽  
Xiang Guo ◽  
Xiaoyi Xu ◽  
Qian Liu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dna Cleavage ◽  
Low Cost ◽  
Nuclease Activity ◽  
Nucleic Acid Detection ◽  
Mesophilic Bacteria ◽  
Brevibacillus Laterosporus ◽  
Double Stranded Dna ◽  
Small Molecule Detection

AbstractThermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

scholarly journals A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

2021 ◽  
Author(s):  
Ekaterina Kropocheva ◽  
Anton Kuzmenko ◽  
Alexei A. Aravin ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
Bacterial Cells ◽  
Dna And Rna ◽  
Rna Targets ◽  
Wide Range ◽  
Mesophilic Bacterium ◽  
Strict Specificity ◽  
Programmable Nucleases

ABSTRACTArgonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

scholarly journals Development of NCOVID-19 Colloidal Gold Immunochromatographic Test Strip

2021 ◽  
Vol 10 (1) ◽  
pp. 1
Author(s):  
Wenyi Liu ◽  
Zhaohua Li
Keyword(s):  
Colloidal Gold ◽  
Low Cost ◽  
Virus Detection ◽  
High Specificity ◽  
Accurate Method ◽  
Rapid Screening ◽  
Nucleic Acid Detection ◽  
Specificity And Sensitivity ◽  
Control Serum

<div><p>Purpose: To establish a fast, simple and accurate method and immunoassay test card for the detection of new coronavirus (nCOVID-19) antigen. Methods: In this study, colloidal gold immunochromatography technology was used to detect nCOVID-19 virus antigens through the sandwich method. At the same time, the preparation plan of colloidal gold was improved, and the application of rapid immune-diagnosis technology in other fields was developed. In this study, purified recombinant nCOVID-19 nucleocapsid protein is used as the antigen to prepare murine monoclonal antibodies. The BN02 antibody produced by the mouse is used as the detection antibody to couple with colloidal gold, forming a gold-labeled complex probe. BN9m1 is used as the coating antibody for the C-line, and ProA is used for the T-line. The polymerization of colloidal gold particles enables us to detect the new coronavirus antigen’s appearance. Thus an in vitro rapid detection kit for virus detection can be made. Results: The positive detection rate of the antigen quality control serum with this colloidal gold reagent was 100%. The specificity was 100%, and the sensitivity was 1ng/ml.  Conclusion: The nCOVID-19 antigen detection reagent (colloidal gold method) developed in this research has high specificity and sensitivity, and can be used in conjunction with nucleic acid detection. As a means of detecting nCOVID-19, it can achieve qualitative and rapid screening of samples with advantage such as accuracy, repeatability, and low cost.</p></div>

A Systematic Review of Current Progresses in the Nucleic Acid-Based Therapies for Neurodegeneration with Implications for Alzheimer’s Disease

2020 ◽  
Vol 20 (15) ◽  
pp. 1499-1517 ◽  
Author(s):  
Maryam Ghaffari ◽  
Nima Sanadgol ◽  
Mohammad Abdollahi
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Neurological Diseases ◽  
Low Cost ◽  
Health Concern ◽  
Systemic Delivery ◽  
Micro Rnas

Recently, manipulation of gene expression and switching genes on or off highlight the potential of nucleic acid-based therapies (NA-BTs). Alzheimer’s Disease (AD) is a common devastating neurodegenerative disease (NDs) responsible for 60-80% of all cases of dementia and predicted as a main public health concern among aged populations. The aim of this study was to outline the current research in the field of NA-BTs for the treatment of AD disabilities, including strategies to suppress the memory and learning defects, to promote recovery processes, and to reinforce social relationships in these patients. This review was performed via evaluating PubMed reported studies from January 2010 to November 2019. Also, reference lists were checked to find additional studies. All intermediation or complementarity of animal models, case-control and cohort studies, and controlled trials (CTs) on specific NA-BTs to AD were acceptable, although in vitro studies were excluded due to the considerable diversities and heterogeneities. After removing the duplicates according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) instruction, we merged remaining titles across search databases. There are 48 ongoing studies related to the application of nucleic acids in the treatment and diagnosis of AD where more consideration is given to DNA targeting strategies (18 targets for vectors and aptamers), antisense oligonucleotides (10 targets), micro-RNAs mimics (7 targets), antagomiRs (6 targets), small interferences-RNAs (5 targets), as well as mRNAs (2 targets) respectively. All of these targets are grouped into 4 categories according to their role in molecular pathways where amyloid-&#946; (18 targets), neural survival (11 targets), memory and cognition (8 targets), and tau (3 targets) are more targeted pathways, respectively. With recent successes in the systemic delivery of nucleic acids via intravenous injection; it is worth investing in the production of new-generation medicines. There are still several challenges for NA-BTs including, their delivery to the effective modulators, mass production at low cost, sustaining efficacy and minimizing off‐target effects. Regarding miRNA-based therapies, given the obvious involvement of miRNAs in numerous facets of brain disease, and the many sophisticated techniques for delivery to the brain, miRNA-based therapies will make new hope for the treatment of neurological diseases such as AD.

Development of an Integrated Instrument for Nucleic Acid Pyrosequencing Detection

2006 ◽  
Vol 315-316 ◽  
pp. 469-473
Author(s):  
Ji Jun Zhu ◽  
H.N. Shi ◽  
J. Cheng ◽  
X.Y. Wei ◽  
Zu Hong Lu
Keyword(s):  
Nucleic Acid ◽  
Personal Computer ◽  
Low Cost ◽  
High Sensitivity ◽  
Structure Design ◽  
Nucleic Acid Detection ◽  
Data Sampling ◽  
Sampling Device ◽  
Automatic Instrument ◽  
Injection Process

This paper introduces a kind of new homemade and low cost multi-channel nucleic acid pyrosequence detector for at least ninety-six channels and presents the detail of related software and hardware development. We construct a kind of automatic instrument to fulfill the pyrosequencing processes. First we select the X-86 personal computer as host computer, the AT89C51 micro-controller as slave computer, the PMT (photoelectric multiply tube) as photoelectric transformation equipment, and the HY-6022 as data sampling device; Second we use the Visual C++ 6.0 as coding tools to design the measure and control system based on Windows 2000 operating system; Third we sample the fluorescent signal in all of the cuvettes during the reaction between nucleic acid and reagent; Last we analyze these data to realize the function of the multi-channel nucleic acid detection. In this paper the whole instrument design and key parts design are both introduced such as the liquid injection process and related structure design, the communication module between the host personal computer and the MCS51, the high sensitivity multi-channel detector (at least 96 channels, the sensitivity is 2.45×10-9w) etc. The result of the instrument for two channels data processing is also reported in this paper.

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