Mediator Displacement Lamp A Novel Method For Universal Sequence Specific Detection Of Isothermal Nucleic Acid Amplification
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Mediator Displacement Lamp A Novel Method For Universal Sequence Specific Detection Of Isothermal Nucleic Acid Amplification
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Author : Lisa Becherer
language : en
Publisher:
Release Date : 2020
Mediator Displacement Lamp A Novel Method For Universal Sequence Specific Detection Of Isothermal Nucleic Acid Amplification written by Lisa Becherer and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with categories.
Abstract: Nucleic acid amplification tests (NAATs) are an essential diagnostic tool throughout the field of life sciences, including clinical applications, food quality control, and environmental monitoring. In the last 20 years, polymerase chain reaction (PCR) has been steadily replaced by isothermal alternatives. These are especially suitable for point-of-need (PON) applications. Loop-mediated isothermal amplification (LAMP) stands out as a particularly robust and highly sensitive method. Special attention is given to the sequence-specific, fluorescence-based detection of LAMP, which allows the highly specific and simultaneous detection of various targets (multiplexing). However, current state-of-the-art methods suffer from complex probe design and elaborate optimization work, which is required for the detection of different targets due to the use of target-specific fluorogenic probes. The principal objectives of this thesis are first, to develop an improved sequence-specific detection method for LAMP with a simplified probe design, second, to verify its analytical performance and third, to validate the method by using it to analyze clinical samples. Furthermore, the feasibility of transferring the novel method to two other platforms, digital nucleic acid testing and electrochemical nucleic acid testing, is investigated. A novel method was successfully developed and named mediator displacement (MD) LAMP. MD LAMP stands out from other state-of-the-art methods because of its use of unique, target-independent fluorogenic reporter molecules. The working principle involves a non-fluorogenic MD probe which features a primer bound to a generic mediator. Mediator displacement occurs during the amplification of target DNA. Fluorescence signal generation is then induced by the interaction between the displaced mediator and a universal reporter molecule. The universal mediator-reporter set can be used to detect various targets. Simplified probe design was demonstrated by the example of a reverse transcription (RT)-LAMP of human immunodeficiency virus (HIV)-1 RNA. The time required for MD probe design was first compared with the time needed to create a state-of-the-art molecular beacon (MD: 10 minutes, molecular beacon: 3-4 hours). Moreover, HIV-1 MD RT-LAMP surpassed molecular beacon-based HIV-1 RT-LAMP, with times to positive 4.1 ± 0.1 minutes shorter (16-20 minutes for 10^3-10^6 HIV RNA copies per reaction) and double the signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, molecular beacon: 2.7 ± 0.4; n = 15). The analytical performance parameters were equally good for both detection methods (limit of detection for MD: 132 HIV RNA copies per reaction, molecular beacon: 141 HIV RNA copies per reaction; linearity for MD and molecular beacon: R^2 = 0.94). Furthermore, multiplex detection of two different targets was demonstrated in a biplex MD RT-LAMP for the simultaneous detection of HIV-1 and human T-lymphotropic virus (HTLV)-1. To present an additional possible application, the biplex RT-LAMP of HIV-1 and HTLV-1 was transferred to a centrifugal microfluidic platform for digital nucleic acid amplification in droplets. Digital NAATs allow precise quantification without the need for standard curves. Compared to existing viral load measurements, the novel assay excelled through the simultaneous and quantitative detection of relevant coinfections. The universal mediator-reporter sets were successfully used for the detection of a second clinically relevant target panel, which is associated with yaws, a neglected tropical disease. For the molecular detection of yaws, a biplex MD LAMP of Treponema pallidum and Haemophilus ducreyi (TPHD-LAMP) was designed, optimized and clinically validated with 293 patient samples. The samples originated from individuals with yaws-like skin lesions in Ghana and Papua New Guinea. So far, no isothermal alternatives to PCR which enable differentiation between T. pallidum, which causes yaws, and H. ducreyi, which is responsible for skin lesions of similar appearance, are available. TPHD-LAMP revealed high diagnostic sensitivities and specificities for T. pallidum (84.7 % and 95.7 %) and H. ducreyi (91.6 % and 84.8 %) compared to TaqMan singleplex qPCR. TPHD-LAMP has become the focus of a large clinical trial conducted in three yaws endemic African countries and linked to the goal of supporting national yaws eradication programmes. So far, the analytical performance of MD LAMP has been verified, the feasibility of a digital MD RT-LAMP has been shown, and MD LAMP has been clinically validated with patient samples. To emphasize the versatility of this novel method, its compatibility with real-time electrochemical NAATs was also demonstrated. Microarray-based electrochemical detection facilitates the simultaneous detection of multiple targets by using spatially-resolved measurement with the help of immobilized probes. In addition, it enables the use of very small and therefore portable devices. To exploit these benefits, MD LAMP was adapted to electrochemical detection by immobilizing universal reporter molecules on an electrode microarray. The feasibility of electrochemical MD LAMP was demonstrated for real-time detection of T. pallidum and end-point detection of HIV-1. Rapid signal increase was observed in real time after 15 minutes for 1000 T. pallidum DNA copies per reaction. Electrochemical MD LAMP enables the use of universal electrode microarrays and could thus bring economic advantages with regard to large-scale fabrication. This thesis concludes with a critical review of the methods and the method-related instrumentation for the sequence-specific detection of LAMP, which are based on a diverse variety of sensing techniques. The multitude of methods is systematically classified and critically evaluated according to a catalogue of criteria covering analytical performance, handling of complex samples, multiplexing, and quantification. The most widespread sensing technique is based on fluorescence detection and is used in around half of the discussed methods. A particular highlight is the universal character of a few fluorescence-based methods, which use generic probes applicable to different targets
Nanobiosensors For Point Of Care Medical Diagnostics
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Author : Manashjit Gogoi
language : en
Publisher: Springer Nature
Release Date : 2023-01-01
Nanobiosensors For Point Of Care Medical Diagnostics written by Manashjit Gogoi and has been published by Springer Nature this book supported file pdf, txt, epub, kindle and other format this book has been release on 2023-01-01 with Medical categories.
This book examines the role of nanobiosensors in point-of-care applications for personalized healthcare and management. It begins by discussing various biomaterials that are used for the development of biosensors in medical diagnostics, and reviews advances in their fabrication and the miniaturization of biosensor devices for lab-on-chip analysis. In turn, it explores the rapidly evolving applications of nanomaterials in the context of biomaterial diagnostics. The book also explores the immense potential of biosensors in medical diagnostics, where they are increasingly being used to detect a wide range of biomolecules and biomarkers. In closing, it discusses the current challenges and outlines the future role of nanobiosensors in the development of next-generation point-of-care applications.
Drugs During Pregnancy And Lactation
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Author : H. Garbis
language : en
Publisher: Elsevier Science
Release Date : 2001-12-21
Drugs During Pregnancy And Lactation written by H. Garbis and has been published by Elsevier Science this book supported file pdf, txt, epub, kindle and other format this book has been release on 2001-12-21 with Medical categories.
The care of pregnant women presents one of the paradoxes of modern medicine. Women usually require little medical intervention during an (uneventful) pregnancy. Conversely, those at high risk of damage to their own health or that of their unborn require the help of appropriate medicinal technology, including drugs. Accordingly, there are two classes of pregnant women, the larger group requires support but not much intervention, while the other needs the full range of diagnostic and therapeutic measures applied in any other branch of medicine. This book presents the current state of knowledge about drugs in pregnancy. In each chapter information is presented separately for two different aspects of the problem seeking a drug appropriate for prescription during pregnancy, and assessing the risk of a drug when exposure has already taken place. Practising clinicians who prescribe medicinal products to women who are, or who may become, pregnant, will find this volume an invaluable reference.
Electrochemical Dna Biosensors
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Author : Mehmet Sengun Ozsoz
language : en
Publisher: CRC Press
Release Date : 2012-04-23
Electrochemical Dna Biosensors written by Mehmet Sengun Ozsoz and has been published by CRC Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-04-23 with Science categories.
This book focuses on the basic electrochemical applications of DNA in various areas, from basic principles to the most recent discoveries. The book comprises theoretical and experimental analysis of various properties of nucleic acids, research methods, and some promising applications. The topics discussed in the book include electrochemical detection of DNA hybridization based on latex/gold nanoparticle and nanotubes; nanomaterial-based electrochemical DNA detection; electrochemical detection of microorganism-based DNA biosensors; gold nanoparticle-based electrochemical DNA biosensors; electrochemical detection of the aptamer-target interaction; nanoparticle-induced catalysis for DNA biosensing; basic terms regarding electrochemical DNA (nucleic acids) biosensors; screen-printed electrodes for electrochemical DNA detection; application of field-effect transistors to label free electrical DNA biosensor arrays; and electrochemical detection of nucleic acids using branched DNA amplifiers.
The Missions Of James Peter And Paul
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Author : Bruce D. Chilton
language : en
Publisher: BRILL
Release Date : 2005-01-01
The Missions Of James Peter And Paul written by Bruce D. Chilton and has been published by BRILL this book supported file pdf, txt, epub, kindle and other format this book has been release on 2005-01-01 with Religion categories.
The Missions of James, Peter, and Paul investigates the nature, diversity, and relationship of three early and important expressions of Judaic Christianity. It is the conviction of the contributors that the Judaic origins of the Christian movement have not been sufficiently understood in both ecclesiastical and academic circles. Comparison with contemporary Judaism is foundational and leads to the question that guides discussion: How did James relate to such prominent figures as Peter and Paul? Given James' own eminence, those relationships must have been hallmarks of his own stance and status, and they open the prospect that we might delineate James' theological perspective more precisely than otherwise possible by means of this contrast with Peter and Paul. That is the reason for the division of the present volume into two parts. The Missions of James, Peter, and Paul is presented in two parts: James and Peter, and James Paul. Several studies investigate the literary and archaeological evidence that clarifies the world in which James, Peter, and Paul lived, while other studies probe exegetical and theological aspects of the discussion.
Molecular Beacons
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Author : Chaoyong James Yang
language : en
Publisher: Springer Science & Business Media
Release Date : 2013-08-15
Molecular Beacons written by Chaoyong James Yang and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-08-15 with Science categories.
Molecular Beacons explains working principle of molecular beacons, discusses their design, synthesis, purification and characterization, explores their thermodynamic and kinetic properties, and more importantly, reviews their in vivo and in vitro applications with the emphasis on the design and modification of molecular beacons for in vivo mRNA imaging applications. This book is designed to bring together in a single resource an organized and comprehensive view of molecular beacons and will be a valuable resource for academic, clinical and industrial scientists and graduate students who may consider exploring molecular beacons in their research or practice. Chaoyong James Yang is the Lu Jiaxi Professor of Chemistry at Xiamen University, China. Weihong Tan is a Distinguished Professor of Chemistry and Biomedical Engineering at Hunan University, China and also a University of Florida Distinguished Professor and V. T. and Louis Jackson Professor of Chemistry at the University of Florida, USA.
Molecular Identification Of Fungi
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Author : Youssuf Gherbawy
language : en
Publisher: Springer Science & Business Media
Release Date : 2010-03-03
Molecular Identification Of Fungi written by Youssuf Gherbawy and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2010-03-03 with Science categories.
Fungi enjoy great popularity in pharmaceutical, agricultural, and biotechnological applications. Recent advances in the decipherment of whole fungal genomes promise an acceleration of these trends. This timely book links scientists from different parts of the world who are interested in the molecular identification of fungi combined with the exploration of the fungal biodiversity in different ecosystems. It provides a compendium for scientists who rely on a rapid and reliable detection of fungal specimens in environmental as well as clinical resources in order to ensure the benefit of industrial and clinical applications. Chapters focus on the opportunities and limits of the molecular marker-mediated identification of fungi. Various methods, procedures and strategies are outlined. Furthermore, the book offers an update of the current progress in the development of fungal molecular techniques, and draws attention to potential and associated problems, as well as integrating theory and practice.
Point Of Care Testing
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Author : Peter Luppa
language : en
Publisher: Springer
Release Date : 2018-07-18
Point Of Care Testing written by Peter Luppa and has been published by Springer this book supported file pdf, txt, epub, kindle and other format this book has been release on 2018-07-18 with Medical categories.
The underlying technology and the range of test parameters available are evolving rapidly. The primary advantage of POCT is the convenience of performing the test close to the patient and the speed at which test results can be obtained, compared to sending a sample to a laboratory and waiting for results to be returned. Thus, a series of clinical applications are possible that can shorten the time for clinical decision-making about additional testing or therapy, as delays are no longer caused by preparation of clinical samples, transport, and central laboratory analysis. Tests in a POC format can now be found for many medical disciplines including endocrinology/diabetes, cardiology, nephrology, critical care, fertility, hematology/coagulation, infectious disease and microbiology, and general health screening. Point-of-care testing (POCT) enables health care personnel to perform clinical laboratory testing near the patient. The idea of conventional and POCT laboratory services presiding within a hospital seems contradictory; yet, they are, in fact, complementary: together POCT and central laboratory are important for the optimal functioning of diagnostic processes. They complement each other, provided that a dedicated POCT coordination integrates the quality assurance of POCT into the overall quality management system of the central laboratory. The motivation of the third edition of the POCT book from Luppa/Junker, which is now also available in English, is to explore and describe clinically relevant analytical techniques, organizational concepts for application and future perspectives of POCT. From descriptions of the opportunities that POCT can provide to the limitations that clinician’s must be cautioned about, this book provides an overview of the many aspects that challenge those who choose to implement POCT. Technologies, clinical applications, networking issues and quality regulations are described as well as a survey of future technologies that are on the future horizon. The editors have spent considerable efforts to update the book in general and to highlight the latest developments, e.g., novel POCT applications of nucleic acid testing for the rapid identification of infectious agents. Of particular note is also that a cross-country comparison of POCT quality rules is being described by a team of international experts in this field.
Viruses And Viral Infections In Developing Countries
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Author : Snežana Jovanović-Ćupić
language : en
Publisher: BoD – Books on Demand
Release Date : 2020-06-17
Viruses And Viral Infections In Developing Countries written by Snežana Jovanović-Ćupić and has been published by BoD – Books on Demand this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020-06-17 with Science categories.
This book contains information on various virus families, with the focus on viruses causing prevalent infections in parts of developing countries in Africa and Asia. Viral proteins play an important role in their replication and infection potential, and are the main candidates for antiviral therapy and vaccines. While some antiviral vaccines are available for quite some time (e.g. MMR), there are regions in the world still struggling with some infections. This is especially the problem in regions where the morbidity rate from viral infections among young children is high. This situation requires urgent measures to put infections under control.
Detection Of Non Amplified Genomic Dna
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Author : Giuseppe Spoto
language : en
Publisher: Springer Science & Business Media
Release Date : 2012-07-06
Detection Of Non Amplified Genomic Dna written by Giuseppe Spoto and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-07-06 with Science categories.
This book offers an overview of state-of-the-art in non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specific detection tools. Advanced materials play multiple roles in ultrasensitive detection. Optical and electrochemical detection tools are among the most widely investigated to analyze non amplified nucleic acids. Biosensors based on piezoelectric crystal have been also used to detect unamplified genomic DNA. The main scientific topics related to DNA diagnostics are discussed by an outstanding set of authors with proven experience in this field.