DNA Methods in Clinical Microbiology

DNA Methods in Clinical Microbiology

DNA Methods Ill Clinical Microbiology by Paul Singleton SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress. ISBN 978-90-481-5456-2 ISBN 978-94-017-1286-6 ( ebook) DOI 10.1007/978-94-017-1286-6 Printed on acid-free paper All Rights Reserved 2000 Springer Science+ Business Media Dordrecht Originally published by Kluwer Academic Publishers in 2000 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

CONTENTS Preface... vii 1 DNA-based technology: an introduction... 1 1.1 The scope of DNA-based methods... 1 1.2 Old versus new technology... 3 1.3 Parameters of DNA-based methods... 4 1.4 DNA-based diagnostic tests... 6 1. 5 'Resolved results'................................................................. 9 2 Nucleic acids: the essentials... 12 2.1 Nucleic acids (RNA and DNA)... 12 2.2 Target sequences... 24 2.3 Specimens... 27 2.4 Extraction of nucleic acids... 30 3 Probe-based (hybridization) methods... 44 3.1 Labelling of probes... 44 3.2 Preparation of samples... 49 3.3 Hybridization (probe-target annealing)... 49 3.4 In situ hybridization... 50 3.5 Examples of probe-based tests... 50 3. 6 Sensitivity and specificity............................................. 53 3. 7 False-positive and false-negative results... 54 4 Nucleic acid amplification I : polymerase chain reaction... 56 4.1 The principle of PCR... 56 4.2 PCR: some practical details... 56 4.3 PCR instrumentation: thermal cyclers... 60 4.4 PCR products: detection and quantification... 61 4.5 PCR inhibitors... 67 4.6 False-positive and false-negative results; controls... 69 4. 7 Contamination with extraneous DNA... 71 4.8 Types of PCR... 73 4. 9 PCR-based studies in clinical microbiology... 83 4.10 Reports from the literature I: detection of pathogens... 84 4.11 Reports from the literature ll: detection of genes... 106 4.12 Reports from the literature m: detection of uncultivable pathogens.. 110 4.13 Reports from the literature N: PCR vs other diagnostic methods... 111 4.14 Reports from the literature V: evaluation of PCR... 118 5 Nucleic acid amplification n: NASBA, TMA, SDA... 126 5.1 NASBA and TMA... 126 5.2 SDA... 131 5.3 Reports from the literature... 138

vi CONTENTS 6 Nucleic acid amplification m: ligase chain reaction... 152 6.1 Thermal cycling... 152 6.2 Extent of amplification... 152 6.3 Detection of products....154 6.4 The Abbott LCx 8 Chlamydia trachomatis assay... 154 6.5 The Abbott LCx 8 Mycobacterium tuberculosis assay... 157 6.6 Anticontamination measures in the LCx assays... 158 6. 7 Reports from the literature (Abbott LCx 8 assays)... 159 7 Nucleic-acid-based typing... 168 7.1 Typing: some applications... 168 7.2 Typing systems: some general characteristics... 170 7.3 Typing: conventional methodology... 171 7.4 Typing: nucleic-acid-based methodology... 172 8 DNA-based ('genotypic') antibiotic susceptibility testing... 203 8.1 Origins of antibiotic resistance... 203 8.2 Antimicrobial susceptibility testing in Chlamydia trachomatis... 205 8.3 Antimicrobial susceptibility testing in Helicobacter pylori... 206 8.4 Antimicrobial susceptibility testing of HIV-1..... 207 8.5 Antimicrobial susceptibility testing of hepatitis B virus... 210 8.6 Antimicrobial susceptibility testing of Staphylococcus aureus... 212 8. 7 Antimicrobial susceptibility testing of Mycobacterium tuberculosis... 217 9 Quantification of pathogens... 229 9.1 bdna... 230 9.2 PCR-based quantification... 232 9.3 NASBA-based quantification... 234 9. 4 Cross-linking quantitative assay... 234 Appendix I The future: chip technology... 237 Appendix n Commercial products, systems and sources... 239 Index... 245

PREFACE For many infectious diseases, diagnosis and management can be facilitated by novel methods based on nucleic acid technology. Some of the methods are in clinical use, while others are currently being evaluated and developed. Unlike gene therapy, and forensic fingerprinting, this technology has not penetrated public awareness; nevertheless, its potential in diagnosis and disease control makes it a valuable addition to clinical microbiology at a critical time when infectious diseases are re-emerging as a major problem. Essentially, pathogens are detected and/or characterized by their unique sequences of nucleotides (subunits in nucleic acids). The target is commonly DNA, but sometimes RNA is examined; 'DNA methods' is used here as a convenient shorthand expression for all methods involving DNA or RNA, or both nucleic acids. This technology is based largely on several recent methods for in vitro amplification of nucleic acids - methods which include not only PCR but also e.g. NASBA, TMA and SDA. Particular methods, adopted by major biotechnology companies, have been rapidly developed for applications which include detection, characterization and/or quantification of a range of pathogens in clinical samples. To these innovations must be added probe-based methods as well as the methods involving signal amplification technology. DNA Methods was written for clinicians, clinical microbiologists, medical students, undergraduates in medical and veterinary microbiology, and those working in clinical laboratories. The text is organized as a concise survey which (i) explains 'molecular' aspects of the methods, and (ii) reports on clinical findings in a number of studies; emphasis is on principle rather than protocol, but detail is included where clarity can be enhanced. No prior knowledge of these methods is assumed; however, while the text starts at the beginning, it includes information published during the preparation of the manuscript, i.e. up to, and including, December 1999. Some of the products cited in the text are currently intended (by the manufacturers) to be used specifically for biomedical research (rather than diagnosis or other clinical purposes). I would like to acknowledge invaluable help from the Medical Library, University of Bristol. I am also grateful to Carolijn van den Reydt and Marlies Vlot (Biomedical Unit, Kluwer Academic Publishers, Dordrecht, The Netherlands) for their assistance in facilitating publication of the book. Paul Singleton Clannaborough Barton Devon, UK January 1st, 2000