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Quantitative detection of water-borne bacterial pathogens by filtration, immunomagnetic separation (IMS) and real-time PCR.

Lui Yuk Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-148). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Abberviations --- p.v / Table of Contents --- p.vii / List of Tables --- p.xiv / List of Figures --- p.xvi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Bacteriological evaluation of water --- p.1 / Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2 / Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3 / Chapter 1.1.3. --- Example of common indicator organisms --- p.3 / Chapter 1.1.3.1. --- Total coliform group --- p.3 / Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4 / Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5 / Chapter 1.1.3.4. --- Klebsiella --- p.5 / Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6 / Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7 / Chapter 1.3.1. --- Bacteria --- p.7 / Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7 / Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11 / Chapter 1.3.1.3 --- Legionella pneumophila --- p.12 / Chapter 1.3.2. --- Protozoa --- p.14 / Chapter 1.3.3. --- Viruses --- p.15 / Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16 / Chapter 1.4.1. --- Examples of conventional detection methods --- p.17 / Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18 / Chapter 1.5. --- Novel approaches for pathogens detection --- p.19 / Chapter 1.5.1. --- Modifications of media --- p.19 / Chapter 1.5.2. --- Antibody-based methods --- p.20 / Chapter 1.5.3. --- Nucleic acid-based methods --- p.21 / Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22 / Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24 / Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26 / Chapter 1.9. --- Aims of this study --- p.28 / Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31 / Chapter 2.1. --- Introduction --- p.31 / Chapter 2.2. --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Bacterial strains --- p.35 / Chapter 2.2.2. --- Bacterial enumeration --- p.35 / Chapter 2.2.3. --- DNA extraction and purification --- p.36 / Chapter 2.2.3.1. --- Boiling method --- p.36 / Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36 / Chapter 2.2.3.3. --- Chelex extraction method --- p.37 / Chapter 2.2.4. --- Targeted sequences --- p.38 / Chapter 2.2.4.1. --- eaeA gene --- p.38 / Chapter 2.2.4.2. --- mdh gene --- p.39 / Chapter 2.2.4.3. --- flaR gene --- p.39 / Chapter 2.2.5. --- PCR amplification --- p.40 / Chapter 2.2.6. --- Gel electrophoresis --- p.41 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Optimization of the PCR --- p.42 / Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42 / Chapter 2.3.2.1. --- Boiling method --- p.42 / Chapter 2.3.2.2. --- Proteinease K method --- p.43 / Chapter 2.3.2.3. --- Chelex method --- p.43 / Chapter 2.3.3. --- Specificity of PCR detection --- p.43 / Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44 / Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44 / Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44 / Chapter 2.4. --- Discussion --- p.57 / Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and Methods --- p.66 / Chapter 3.2.1. --- Bacterial strains --- p.66 / Chapter 3.2.2. --- Bacterial enumeration --- p.66 / Chapter 3.2.3. --- Filtration --- p.67 / Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68 / Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68 / Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68 / Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70 / Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70 / Chapter 3.2.6. --- DNA extraction --- p.71 / Chapter 3.2.7. --- Multiplex PCR --- p.71 / Chapter 3.2.8. --- PCR amplification --- p.72 / Chapter 3.2.9. --- Gel electrophoresis --- p.72 / Chapter 3.3. --- Results --- p.73 / Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2. --- Detection limit of PCR --- p.73 / Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2.2. --- Influence of background flora --- p.73 / Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77 / Chapter 3.3.3. --- Multiplex PCR --- p.77 / Chapter 3.4. --- Discussion --- p.91 / Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94 / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Materials and Methods --- p.99 / Chapter 4.2.1. --- Bacteria strains --- p.99 / Chapter 4.2.2. --- Bacterial enumeration --- p.99 / Chapter 4.2.3. --- Primers and Probes --- p.100 / Chapter 4.2.3.1. --- eaeA gene --- p.101 / Chapter 4.2.3.2. --- mdh gene --- p.102 / Chapter 4.2.3.3. --- flaR gene --- p.102 / Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103 / Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103 / Chapter 4.2.4.2. --- Purification of PCR product --- p.104 / Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105 / Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105 / Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106 / Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107 / Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108 / Chapter 4.3. --- Results --- p.110 / Chapter 4.3.1. --- Determination of targeted sequences --- p.110 / Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110 / Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114 / Chapter 4.3.4. --- Specificity of real-time PCR --- p.121 / Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121 / Chapter 4.4. --- Discussion --- p.131 / Chapter 5. --- Conclusion and future perspectives --- p.133 / Chapter 6. --- References --- p.138

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323531
Date January 2001
ContributorsLui, Yuk Sun., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xix, 148 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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