by Lap-Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 90-101). / Abstract also in Chinese. / Abstract --- p.i / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- The biology of hepatitis A virus --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- General characteristics of HAV --- p.2 / Chapter 1.1.3 --- Stability and disinfection of HAV --- p.3 / Chapter 1.1.4 --- Molecular biology of HAV --- p.5 / Chapter 1.1.4.1 --- Genomic organization of HAV --- p.5 / Chapter 1.1.4.2 --- Antigenic sites on the capsid of HAV --- p.8 / Chapter 1.1.5 --- Laboratory diagnosis and methods of study for HAV --- p.8 / Chapter 1.1.5.1 --- Cell-culture propagation and antigen detection --- p.8 / Chapter 1.1.5.2 --- Nucleic acid detection --- p.10 / Chapter 1.1.6 --- Epidemiology of HAV --- p.12 / Chapter 1.1.6.1 --- Distribution of HAV infection --- p.12 / Chapter 1.1.6.2 --- Seasonal pattern of HAV infection --- p.13 / Chapter 1.1.6.3 --- Mode of transmission --- p.13 / Chapter 1.1.6.4 --- Molecular epidemiology --- p.15 / Chapter 1.1.7 --- Epidemiology of HAV infection in Hong Kong --- p.15 / Chapter 1.2 --- Transmission of viruses through contaminated food --- p.18 / Chapter 1.2.1 --- Active accumulation of water contaminants by shellfish --- p.20 / Chapter 1.2.2 --- Retention of viruses by shellfish in contaminate water --- p.21 / Chapter 1.2.3 --- Elimination of viruses in contaminated shellfish --- p.21 / Chapter 1.2.4 --- Indicators for contamination by enteric viruses --- p.21 / Chapter 1.3 --- Detection of viruses from foods --- p.23 / Chapter 1.3.1 --- Recovery of viruses from foods --- p.23 / Chapter 1.3.2 --- Detection of viral nucleic acid --- p.23 / Chapter 1.4 --- Objectives of the study --- p.26 / Chapter Chapter 2- --- Materials and methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Sera from patients with acute viral hepatitis --- p.27 / Chapter 2.1.2 --- Collection of shellfish samples --- p.28 / Chapter 2.1.3 --- Purified HAV preparations as positive control --- p.30 / Chapter 2.1.4 --- Control samples for the virion capture method --- p.30 / Chapter 2.1.5 --- Preparation of dissecting instruments and processing of shellfish samples --- p.30 / Chapter 2.1.6 --- Plasticwares and glasswares --- p.30 / Chapter 2.1.7 --- "Chemicals, reagents and commercial kits" --- p.31 / Chapter 2.1.7.1 --- Samples processing --- p.31 / Chapter 2.1.7.2 --- Reagents for RNA extractions --- p.31 / Chapter 2.1.7.3 --- Oligonucleotide primers synthesis --- p.33 / Chapter 2.1.7.4 --- Primers purification after synthesis --- p.34 / Chapter 2.1.7.5 --- Gel electrophoresis --- p.34 / Chapter 2.1.7.6 --- Reagents for hybridization --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Samples processing --- p.37 / Chapter 2.2.2 --- Artificially seeded HAV in shellfish --- p.37 / Chapter 2.2.3 --- RNA extraction methods --- p.37 / Chapter 2.2.3.1 --- Acid phenol method --- p.37 / Chapter 2.2.3.2 --- Spin cartridge method --- p.38 / Chapter 2.2.3.3 --- Virion capture method --- p.39 / Chapter 2.2.4 --- "Oligonucleotides used for RT, PCR and hybridization" --- p.40 / Chapter 2.2.4.1 --- Oligonucleotides used in HAV RT-PCR --- p.40 / Chapter 2.2.4.2 --- Primer set used for the evaluation of inhibitors of PCR in shellfish homogenates --- p.41 / Chapter 2.2.4.3 --- Preparation of oligonucleotide primers --- p.41 / Chapter 2.2.4.4 --- Detachment of the primer from the column --- p.42 / Chapter 2.2.4.5 --- Purification of the oligonucleotides --- p.42 / Chapter 2.2.4.6 --- Confirmation of synthesed oligonucleotide --- p.43 / Chapter 2.2.5 --- Reverse transcription of HAV genomic RNA template and PCR --- p.43 / Chapter 2.2.6 --- Human β-actin gene PCR for the evaluation of shellfish homogenates --- p.45 / Chapter 2.2.7 --- Analysis of PCR products --- p.46 / Chapter 2.2.7.1 --- Agarose gel electrophoresis for the analysis of PCR products --- p.46 / Chapter 2.2.7.2 --- Dot blot hybridization for the confirmation of PCR products --- p.46 / Chapter 2.2.7.3 --- Southern blot hybridization for the confirmation of PCR products --- p.47 / Chapter 2.2.7.4 --- 5'-end DNA labelling of oligonucleotide probe --- p.47 / Chapter 2.2.7.5 --- Hybridization in sodium chloride / sodium citrate --- p.48 / Chapter Chapter 3- --- Results / Chapter 3.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.52 / Chapter 3.2 --- Synthesis and yields of oligonucleotide primers --- p.54 / Chapter 3.3 --- Development of reverse-transcription polymerase chain reaction (RT-PCR) for HAV --- p.55 / Chapter 3.4 --- Sampling of shellfish from different markets in Hong Kong --- p.59 / Chapter 3.5 --- Quantitation of HAV RNA in stock virus preparations --- p.62 / Chapter 3.6 --- Comparison of RNA extraction methods and the detection limit of the established RT-PCR method for HAV --- p.62 / Chapter 3.7 --- Specificity of the RT-PCR in combination with virion capture method for the detection of HAV --- p.65 / Chapter 3.8 --- Detection of HAV RNA by RT-PCR in shellfish in Hong Kong / Chapter Chapter 4- --- Discussion / Chapter 4.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.76 / Chapter 4.2 --- Development of RT-PCR method for the detection of HAV --- p.78 / Chapter 4.3 --- Evaluation of RNA extraction methods for the detection of HAV in shellfish sample by RT-PCR --- p.78 / Chapter 4.4 --- Application of the established RT-PCR method for the detection of HAV contamination in locally available shellfish --- p.79 / Chapter Chapter 5- --- References --- p.90 / Appendix I Figures of agarose gel electrophoresis of RT-PCR products for all samples (I1 -I23 ) --- p.101 / Appendix II Figures of dot-blot hybridization assay (II2 -II4 ) --- p.114
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322501 |
| Date | January 1998 |
| Contributors | Lam, Lap-Yee., Chinese University of Hong Kong Graduate School. Division of Microbiology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xii, 117 leaves : ill. (some col., some mounted) ; 30 cm. |
| Rights | Use 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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