Real-time PCR in Microbiology

Real-time PCR in Microbiology PDF

Author:

Publisher:

Published: 2007

Total Pages:

ISBN-13: 9781913652357

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The editor and authors have produced an excellent book that will be extremely useful for all microbiologists. A recommended book for all microbiology laboratories.

Gene Quantification

Gene Quantification PDF

Author: Francois Ferre

Publisher: Springer Science & Business Media

Published: 2012-12-06

Total Pages: 379

ISBN-13: 1461241642

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Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur and Doty (1961) first showed that denaturation of the double helix could be reversed - that the process of molecular reassociation was exquisitely sequence dependent. Gillespie and Spiegelman (1965) developed a way of using the method to titrate the number of copies of a probe within a target sequence in which the target sequence was fixed to a membrane support prior to hybridization with the probe - typically a RNA. Thus, this was a precursor to many of the methods still in use, and indeed under development, today. Early examples of the application of these methods included the measurement of the copy numbers in gene families such as the ribosomal genes and the immunoglo bulin family. Amplification of genes in tumors and in response to drug treatment was discovered by this method. In the same period, methods were invented for estimating gene num bers based on the kinetics of the reassociation process - the so-called Cot analysis. This method, which exploits the dependence of the rate of reassociation on the concentration of the two strands, revealed the presence of repeated sequences in the DNA of higher eukaryotes (Britten and Kohne, 1968). An adaptation to RNA, Rot analysis (Melli and Bishop, 1969), was used to measure the abundance of RNAs in a mixed population.

The Polymerase Chain Reaction

The Polymerase Chain Reaction PDF

Author: Kary B. Mullis

Publisher: Springer Science & Business Media

Published: 2012-02-02

Total Pages: 464

ISBN-13: 1461202574

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James D. Watson When, in late March of 1953, Francis Crick and I came to write the first Nature paper describing the double helical structure of the DNA molecule, Francis had wanted to include a lengthy discussion of the genetic implications of a molecule whose struc ture we had divined from a minimum of experimental data and on theoretical argu ments based on physical principles. But I felt that this might be tempting fate, given that we had not yet seen the detailed evidence from King's College. Nevertheless, we reached a compromise and decided to include a sentence that pointed to the biological significance of the molecule's key feature-the complementary pairing of the bases. "It has not escaped our notice," Francis wrote, "that the specific pairing that we have postulated immediately suggests a possible copying mechanism for the genetic material." By May, when we were writing the second Nature paper, I was more confident that the proposed structure was at the very least substantially correct, so that this second paper contains a discussion of molecular self-duplication using templates or molds. We pointed out that, as a consequence of base pairing, a DNA molecule has two chains that are complementary to each other. Each chain could then act ". . . as a template for the formation on itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before" and, moreover, " ...

PCR Detection of Microbial Pathogens

PCR Detection of Microbial Pathogens PDF

Author: Mark Wilks

Publisher: Humana Press

Published: 2016-08-23

Total Pages: 317

ISBN-13: 9781493960996

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PCR methods for the detection of microbial pathogens have made relatively little impact in diagnostic microbiology laboratories due to the common decision to use expensive commercially produced tests rather than the cheaper alternative of developing one’s own tests or introducing tests developed by other workers. PCR Detection of Microbial Pathogens, Second Edition presents alternatives to commercially produced PCR methods to detect microbial pathogens. Although most of the chapters in this book are devoted to the detection of specific pathogens, the first chapters in this book should appeal to anyone working in this field regardless of their particular interests. Although PCR tests can often be made to work with relatively little effort, it is often unclear how efficient the PCR test is, how inhibitory the specimen containing the pathogen of interest is and how the test can be quality controlled. All of which are of great importance in developing tests for diagnostic use. These topics are covered in great depth at the beginning of the book. The main part of the book is devoted to describing methods for the detection of a wide range of pathogens and from widely different specimens and situations. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, PCR Detection of Microbial Pathogens, Second Edition serves microbiologists regardless of their particular interest because, when used together with the general principles, the sheer variety of procedures provided here enables the reader to design and introduce diagnostic tests in the laboratory with confidence.

Real-time PCR

Real-time PCR PDF

Author: M Dorak

Publisher: Garland Science

Published: 2007-01-24

Total Pages: 484

ISBN-13: 1134183992

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With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.

PCR Detection of Microbial Pathogens

PCR Detection of Microbial Pathogens PDF

Author: Konrad Sachse

Publisher: Springer Science & Business Media

Published: 2003

Total Pages: 667

ISBN-13: 1588290492

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Hands-on laboratory experts present a set of "classic" PCR-based methods for the identification and detection of important animal and food microbial pathogens, including several zoonotic agents. These proven techniques can be precisely applied to a wide variety of microbes, among them Campylobacter spp., chlamydiae, toxigenic clostridia, Escherichia coli (STEC), Listeria monocytogenes, mycoplasmas, salmonellae, and Yersinia enterocolitica. Additional chapters review the specificity and performance of diagnostic PCR analysis, the pre-PCR processing of samples, the critical aspects of standardizing PCR methods, and the general issues involved in using PCR technology for microbial diagnosis.

Rapid Cycle Real-Time PCR — Methods and Applications

Rapid Cycle Real-Time PCR — Methods and Applications PDF

Author: W. Dietmaier

Publisher: Springer Science & Business Media

Published: 2013-06-29

Total Pages: 200

ISBN-13: 3642593976

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Rapid-Cycle Real-Time PCR is a powerful technique for nucleic acid amplification and analysis that often requires less than half an hour to perform. Samples are amplified by rapid-cycle PCR followed by immediate melting curve analysis in the same instrument. Melting curve analysis of PCR products with SYBR Green I often allows product identification without gel electrophoresis. Furthermore, in the presence of fluorescent hybridization probes, melting curves provide "dynamic dot blots" for fine sequence analysis, including single nucleotide polymorphisms (SNPs). The method is often cited as the most versatile, efficient method for nucleic acid analysis in research and diagnostics in the fields of genetics and oncology. Molecular diagnostics has never been easier!

Rapid Detection and Identification of Infectious Agents

Rapid Detection and Identification of Infectious Agents PDF

Author: David Kingsbury

Publisher: Elsevier

Published: 2012-12-02

Total Pages: 309

ISBN-13: 0323156576

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Rapid Detection and Identification of Infectious Agents is a collection of papers presented at the International Symposium on Rapid Detection and Identification of Infectious Agents held on October 5-7, 1983, in Oakland, California, and organized by the Naval Biosciences Laboratory of the School of Public Health of the University of California at Berkeley. Contributors examine progress in the field of rapid diagnosis of infectious diseases, with a particular emphasis on DNA probe-based assays and monoclonal and polyclonal antibody-based immunoassays. This volume is organized into five sections encompassing 20 chapters. It begins with an overview of state-of-the-art methods for rapid detection and identification of infectious agents, including technology that is currently applied in clinical microbiology, as well as concerns regarding the political and scientific climates, which have an impact on health care and clinical microbiology. Chapters are organized to deal with a single diagnostic type of test for a given broad group of organisms. The approach is to compare the strengths and weaknesses of each of the new diagnostic procedures, using the same type of clinical material whenever possible. The book gives consideration to the fundamental design of DNA probes and probe assay systems, the clinical comparison of immunologic assays for the diagnosis of meningococcal disease, and immunodiagnostics for viral and parasitic pathogens. This book will be of value to scientists and researchers interested in immunology and infectious diseases, as well as the methods used to detect and identify them.

Molecular Microbial Ecology

Molecular Microbial Ecology PDF

Author: Mark Osborn

Publisher: Taylor & Francis

Published: 2004-06-02

Total Pages: 256

ISBN-13: 1135320411

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Microoganisms are distributed across every ecosystem, and microbial transformations are fundamental to the operation of the biosphere. Microbial ecology is the study of this interaction between microorganisms and their environment, and arguably represents one of the most important areas of biological research. Yet for many years our study of microbial flora was severely limited: the primary method of culturing microorganisms on media allowed us to study only between 0.1 and 10% of the total microbial flora in any given environment. Molecular Microbial Ecology gives a comprehensive guide to the recent revolution in the study of microorganisms in the environment. Details are given on molecular methods for isolating some of the previously uncultured and numerically dominant microbial groups. PCR-based approaches to studying prokaryotic systematics are described, including ribosomal RNA analysis and stable isotope probing. Later chapters cover DNA hybridisation techniques (including fluorescent in situ hybridisation), as well as genomic and metagenomic approaches to microbial ecology. Gathering together some of the world’s leading experts, this book provides an invaluable introduction to the modern theory and molecular methods used in studying microbial ecology.