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Your essential guide to the design, operation, management, and health care integration of the modern molecular microbiology laboratory.
This comprehensive resource offers definitive guidance on the operational and interpretive aspects of clinical molecular microbiology. Tailored for medical laboratory professionals, it provides practical “how-to” guidance for establishing, maintaining, and advancing molecular microbiology testing services and details the unique expertise required to support infectious disease diagnostics.
The Manual offers a clear and practical roadmap for topics ranging from selecting appropriate technologies, instruments, and analytic pipelines to navigating complex interpretive challenges and positioning diagnostic testing services for future clinical and population health needs.
Beginning with foundational technologies and their clinical applications, this book offers accessible overviews of each method's potential, implications, and emerging roles. Subsequent sections dive meticulously into details of laboratory setup, design, and operations, empowering readers with hands-on insights for routine and advanced testing methods, including advanced sequencing technologies. It also tackles the nuanced challenges of interpreting and reporting results from cutting-edge diagnostics, including those focused on antimicrobial resistance and metagenomics.
The final section explores the broader impact of molecular microbiology on value-based care, with discussions on clinical management, laboratory stewardship, and the future of molecular diagnostics in public health.
Comprehensive and forward-looking, the Manual of Molecular Microbiology equips readers with both foundational knowledge and practical expertise, making it an indispensable reference for today's clinical laboratory professionals.
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Cover
Table of Contents
Title Page
Copyright Page
Contributors
Preface
Author and Editor Conflicts of Interest
section I: Technologies and Clinical Applications
1 Real‐Time PCR
INTRODUCTION
REAL‐TIME PCR
BRIEF ASSAY DESIGN CONSIDERATIONS
VARIATIONS ON REAL‐TIME PCR
ADVANTAGES AND LIMITATIONS OF REAL‐TIME PCR
CLINICAL APPLICATIONS OF REAL‐TIME PCR
SUMMARY
REFERENCES
2 Digital PCR
INTRODUCTION TO DIGITAL PCR
SPECIFIC EXPERIMENTAL CONSIDERATIONS FOR dPCR
QUANTIFICATION BY dPCR
SOURCES OF ERROR
THE ABILITY OF dPCR TO PERFORM ACCURATE QUANTIFICATION
FURTHER CONSIDERATIONS FOR RNA TARGETS
THE dMIQE GUIDELINES
POTENTIAL APPLICATIONS OF dPCR TO MICROBIAL RESEARCH AND DIAGNOSTICS
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
3 Isothermal Nucleic Acid Amplification Technologies and CRISPR‐Cas‐Based Nucleic Acid Detection Strategies for Infectious Diseases Diagnostics
INTRODUCTION
LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION
TRANSCRIPTION‐MEDIATED AMPLIFICATION
OTHER ISOTHERMAL NAA TECHNOLOGIES: HELICASE‐DEPENDENT AMPLIFICATION AND STRAND DISPLACEMENT AMPLIFICATION
CRISPR‐CAS SYSTEMS
CONCLUSIONS AND PERSPECTIVES
ACKNOWLEDGMENTS
REFERENCES
4 Strain Typing (Bacterial, Viral, Fungal, and Mycobacterial)
INTRODUCTION TO STRAIN TYPING
IMPORTANCE OF STRAIN TYPING
BACTERIAL STRAIN TYPING
VIRAL STRAIN TYPING
FUNGAL STRAIN TYPING
METHODOLOGIC CHALLENGES
STANDARDIZATION
PREANALYTICAL CONSIDERATIONS
ANALYTICAL CONSIDERATIONS
POSTANALYTICAL PROCESSES
THE FUTURE OF STRAIN TYPING IN THE ERA OF GLOBAL SURVEILLANCE
REFERENCES
5 Antimicrobial Resistance Genotyping
INTRODUCTION
METHODS FOR ANTIMICROBIAL RESISTANCE TESTING
VIRAL AMR GENOTYPING
BACTERIAL AMR GENOTYPING
FUNGAL AMR GENOTYPING
SUMMARY
ACKNOWLEDGMENTS
REFERENCES
6 Broad‐Range Diagnostic Amplification and Detection
BACKGROUND
PHYLOGENETIC PROPERTIES AND LIMITATIONS OF THE 16S RRNA GENE
SELECTING PRIMERS FOR 16S RRNA BROAD‐RANGE AMPLIFICATION FROM CLINICAL SAMPLES
BROAD‐RANGE PCR
RELEVANT SAMPLE TYPES FOR BROAD‐RANGE AMPLIFICATION
BROAD‐RANGE AMPLIFICATION AND SANGER SEQUENCING
BROAD‐RANGE AMPLIFICATION AND NEXT‐GENERATION SEQUENCING
BROAD‐RANGE AMPLIFICATION AND LONG‐READ SEQUENCING
BROAD‐RANGE AMPLIFICATION OF ALTERNATIVE TARGETS
CLINICAL RELEVANCE OF BROAD‐RANGE AMPLIFICATION
PERSISTENT BACTERIAL DNA
IS DETECTION OF ALL SPECIES NECESSARY?
IS DETECTION SUFFICIENT?
SEQUENCING TECHNOLOGIES AND TIMELY RESULTS
BROAD‐RANGE AMPLIFICATION AND DETECTION OF FUNGI
COMPETING APPROACHES
CONCLUSIONS
REFERENCES
7 Characterization of the Human Microbiome in Health and Disease
OVERVIEW OF THE HUMAN MICROBIOME
THE HYGIENE HYPOTHESIS
THE ROLE OF THE MICROBIOTA IN HUMAN HEALTH
TRANSLATING MICROBIOME SCIENCE TO THE CLINIC
CONCLUDING THOUGHTS
REFERENCES
8 Host Transcriptomics: Clinical Applications
INTRODUCTION
TRANSCRIPTOMICS
REFERENCES
9 Organism Transcriptomics
TRANSCRIPTOMICS: GENERAL
BRIEF HISTORY OF TECHNOLOGY
BENEFITS OF PATHOGEN TRANSCRIPTOMICS
CURRENT STATE‐OF‐THE‐ART TECHNIQUES
PATHOGEN SINGLE‐CELL RNA‐SEQ APPLICATIONS
CHALLENGES AND FUTURE DIRECTIONS
CONCLUSION
REFERENCES
section II: Design, Implementation, and Operations: General
10 Laboratory Design for Molecular Technologies
INTRODUCTION
CONSIDERATIONS FOR MINIMIZING NUCLEIC ACID CONTAMINATION
THE SIGNIFICANCE OF EQUIPMENT IN LABORATORY DESIGN
LEAN LABORATORY DESIGN
SUMMARY
REFERENCES
11 Sample Selection, Preservation, Transport, and Storage
INTRODUCTION
SPECIMEN SELECTION
SPECIMEN COLLECTION CONSIDERATIONS
TRANSPORT MEDIUM
TRANSPORT REGULATIONS
SPECIMEN PROCESSING AND STORAGE
COLLECTION METHODS AND PROCESSING OF SELECTED SPECIMENS
SUMMARY
REFERENCES
12 Nucleic Acid Extraction and Purity, Including Performance Considerations of Different Extraction Chemistries
INTRODUCTION
OVERVIEW OF EXTRACTION
LIQUID‐PHASE EXTRACTION
SOLID‐PHASE EXTRACTION
COMMERCIALLY AVAILABLE EXTRACTION SYSTEMS
AUTOMATION IN EXTRACTION
EXTRACTION AND SAMPLE‐TO‐ANSWER TESTING
EXTRACTION‐FREE APPROACHES
ADOPTION OF EXTRACTION TECHNIQUES
EXTRACTION CHALLENGES
ASSESSING PURITY AND PERFORMANCE
ONGOING ASSESSMENT OF PERFORMANCE
SUMMARY
REFERENCES
13 Selection of Nucleic Acid Amplification Systems:
In Vitro
Diagnostic Tests
INTRODUCTION
DEFINING THE NEED
EXPLORING POTENTIAL IVD SOLUTIONS
CLOSING
REFERENCES
APPENDIX A Clinical microbiology new test request form
APPENDIX B Checklist for selection of nucleic acid amplification IVD system
14 Selection of Nucleic Acid Amplification Systems: Laboratory Developed Tests
INTRODUCTION
REGULATION OF LDTs
DESIGNING AN LDT
DESIGNING THE MOLECULAR REACTION
CONCLUSION
REFERENCES
15 Verification, Validation, and Quality Assurance for Nucleic Acid Amplification Tests
DEFINING VERIFICATION AND VALIDATION: AN OVERVIEW
STUDY DESIGN
DATA ANALYSIS
ONGOING QUALITY ASSURANCE
CONCLUSIONS
REFERENCES
16 Molecular Microbiology Standardization
INTRODUCTION
DEFINITION OF TERMS
TRACEABILITY OF STANDARDS
ISO 17511:2020
QUANTITATIVE AND DIGITAL PCR
COMPOSITION OF THE REFERENCE MATERIAL
COMMUTABILITY
CONCLUSION
REFERENCES
section III: Design, Implementation, and Operations: Sequencing‐Based Technologies
17 Sequencing Platforms
INTRODUCTION
SEQUENCING TECHNOLOGIES
AUTOMATION
SELECTING THE RIGHT SEQUENCING PLATFORM
REFERENCES
18 Nucleic Acid Extraction and Next‐Generation Sequencing Library Prep
INTRODUCTION
DNA/RNA EXTRACTION
LIBRARY PREPARATION
CONCLUDING SUMMARY
ACKNOWLEDGMENTS
REFERENCES
19 Considerations of Depth, Coverage, and Other Read Quality Metrics
INTRODUCTION
READ QUALITY
READ PROCESSING
READ ASSEMBLY
READ MAPPING
SPECIALIZED APPROACHES AND REPRODUCIBILITY
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
20 Pipelines and Databases—Genome Assembly and Analysis from Bacterial Isolates
INTRODUCTION
FROM READS TO ANALYSIS RESULTS
CONCLUSION
REFERENCES
21 Analysis of Metagenomic Next‐Generation Sequencing Data Obtained from Clinical Samples
INTRODUCTION
ASSAY WORKFLOW
DATA PROCESSING PIPELINES
GENOMIC AND TAXONOMIC DATABASES
INTERPRETATION
IMPLEMENTATION CONSIDERATIONS
CONCLUSION
REFERENCES
22 Pipelines and Databases—Microbiome Analysis
METAGENOMICS DATA PROCESSING
ASSEMBLY AND BINNING
TAXONOMIC PROFILING
METAGENOME POPULATION ANALYSIS
DIFFERENTIAL ABUNDANCE ANALYSIS
CONCLUSION
REFERENCES
23 Validation and Quality Control of Next‐Generation Sequencing Assays
BACKGROUND AND SCOPE
FAMILIARIZATION AND PLANNING
QUALITY CONTROL (QC)
VALIDATION AND VERIFICATION
QUALITY CONTROL AND ASSURANCE
ACKNOWLEDGMENTS
REFERENCES
24 Application‐Specific Considerations for Clinical and Epidemiologic Sequencing
INTRODUCTION
OVERVIEW OF SEQUENCING APPROACHES AND IMPACT ON DOWNSTREAM ANALYSES
PATIENT MANAGEMENT APPLICATIONS
HOSPITAL/PUBLIC HEALTH APPLICATIONS
PRACTICAL CONSIDERATIONS FOR QUALITY CONTROL
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
25 Generation and Analysis of Host Transcriptomics and Development of Host‐Response Based Signatures
INTRODUCTION
CONSIDERATIONS FOR STUDY DESIGN
LABORATORY APPROACH TO TRANSCRIPTIONAL PROFILING
COMPUTATIONAL APPROACH TO TRANSCRIPTOMIC ANALYSIS
CLINICAL TRANSLATION AND APPLICATION OF HOST TRANSCRIPTIONAL SIGNATURES
REFERENCES
section IV: Special Challenges in Result Interpretation and Reporting
26 Bacterial Antimicrobial Susceptibility Testing
INTRODUCTION
GENOTYPIC TESTS FOR ANTIMICROBIAL RESISTANCE AND SUSCEPTIBILITY
INTERPRETATION
CONCLUSIONS
REFERENCES
27 Public Health Surveillance
PUBLIC HEALTH LABORATORY AND SURVEILLANCE OVERVIEW
NATIONAL PUBLIC HEALTH SURVEILLANCE PROGRAMS
CONCLUSION
REFERENCES
28 Considerations in Molecular Strain Typing for Bacteria, Fungi, and Viruses
INTRODUCTION
SPECIAL CONSIDERATIONS FOR BACTERIA
SPECIAL CONSIDERATIONS FOR FUNGI
SPECIAL CONSIDERATIONS FOR VIRUSES
EMERGING TECHNIQUES AND BEST PRACTICES
CONCLUSION
REFERENCES
29 Considerations in Result Interpretation and Reporting for Untargeted and Broadly Targeted Pathogen Detection Sequencing Assays
INTRODUCTION
CONCLUSION
REFERENCES
30 Considerations in Result Interpretation and Reporting for Microbiome Analysis
INTRODUCTION
REGULATORY CONSIDERATIONS
CLINICAL RELEVANCE OF MICROBIOME DATA
TEST DEVELOPMENT CONSIDERATIONS
TRANSLATING MICROBIOME RESEARCH
OUTLINE FOR ADVANCING MICROBIOME‐BASED MEDICINE
OUTLOOK FOR THE FUTURE
REFERENCES
31 Clinical Interpretation of Ultrasensitive Molecular Assays
INTRODUCTION
MOLECULAR TEST METHODS
INFECTIONS NOT ASSOCIATED WITH COLONIZATION
COLONIZATION
PERSISTENCE, SHEDDING, AND VACCINE
CONCLUSION
REFERENCES
section V: Molecular Microbiology’s Impact on Value‐based Care
32 Clinical Interpretation and Diagnostic Management
INTRODUCTION
SAMPLE COLLECTION
SYNDROMIC PANELS AND INTERPRETATION
PATHOGEN‐SPECIFIC TESTS AND INTERPRETATION
QUALITATIVE AND QUANTITATIVE TESTS
NEXT‐GENERATION SEQUENCING
SUMMARY
REFERENCES
33 The Role of Clinical Microbiology in Antimicrobial and Diagnostic Stewardship
INTRODUCTION
ANTIMICROBIAL RESISTANCE (AMR)
GENERAL TESTING VALUE: CONSIDERATIONS AND PERSPECTIVES
MOLECULAR TEST MENU OPTIONS AND CONCEPTS
ANTIMICROBIAL STEWARDSHIP AS A CONTEXT FOR DIAGNOSTICS
LOGISTIC CONSIDERATIONS FOR DEPLOYING RAPID MOLECULAR DIAGNOSTICS
DIAGNOSTIC STEWARDSHIP LOGISTICS TO IMPROVE ANTIMICROBIAL PRESCRIBING
REFERENCES
34 Evidence‐Based Assay Deployment—Support of Population Health Initiatives
INTRODUCTION AND CONCEPTS
CONTRIBUTIONS OF MOLECULAR MICROBIOLOGY METHODS TO POPULATION HEALTH
MOLECULAR MICROBIOLOGY AND POPULATION HEALTH BEYOND CLINICAL CARE
NEW INITIATIVES FOR PUBLIC HEALTH: WASTEWATER MONITORING AND WASTEWATER NETWORKS
MOLECULAR MICROBIOLOGY FOR POPULATION HEALTH SURVEILLANCE, OUTBREAK DETECTION, AND DISEASE CONTROL
PORTABLE MOLECULAR MICROBIOLOGY ASSAYS: MOBILE MICROBIOLOGY GOES TO UNDERSERVED POPULATIONS
INFRASTRUCTURE REQUIRED FOR EFFECTIVE DEPLOYMENT OF MOLECULAR MICROBIOLOGY FOR POPULATION HEALTH
THE ROLE OF CLINICAL LABORATORIES
MOLECULAR MICROBIOLOGY AND THE SOCIAL DETERMINATES OF HEALTH (SDOH)
UNMET NEEDS FOR MOLECULAR MICROBIOLOGY DIAGNOSTICS FOR POPULATION HEALTH
SUMMARY
REFERENCES
35 Evidence‐Based Assay Deployment—Precision Medicine Initiatives
INTRODUCTION
APPLYING EVIDENCE‐BASED DEPLOYMENT STRATEGIES
CHALLENGES IN EVIDENCE‐BASED DEPLOYMENT
INTEGRATING EVIDENCE‐BASED DEPLOYMENT INTO VALUE‐BASED CARE (VBC)
REFERENCES
36 Evidence‐Based Assay Deployment—Infrastructure, Documentation, and Strategy
INTRODUCTION
MOLECULAR MICROBIOLOGY LABORATORIES CAN CONTRIBUTE TO VALUE‐BASED CARE (VBC)
POPULATION HEALTH: SERVING SUBPOPULATIONS WITH PREVENTIVE CARE AND WELLNESS
BARRIERS TO VBC
CONDUCTING PATIENT‐ORIENTED RESEARCH (POR)
CHECKLISTS AND GUIDELINES TO IMPROVE EXPERIMENTAL DESIGN AND PUBLICATIONS FOR INCLUSION INTO EVIDENCE‐BASED AND QUALITY GUIDELINES FOR VBC
ADDITIONAL SKILLS AND INFRASTRUCTURE
DOCUMENTING LABORATORY IMPACT
STRATEGIC THINKING
CONCLUSIONS
REFERENCES
Index
End User License Agreement
Chapter 1
TABLE 1 Examples of commercially available DNA intercalating dyes used for ...
TABLE 2 Select common dyes used for real‐time PCR
TABLE 3 Clinical indications with CLIA‐waived PCR tests
a
TABLE 4 Select FDA‐cleared high‐throughput real‐time PCR platforms
Chapter 2
TABLE 1 Details of the currently available dPCR instruments
Chapter 3
TABLE 1 Commercially available, FDA‐cleared/approved or CE‐marked LAMP‐base...
TABLE 2 Examples of commercially available, FDA‐cleared/approved TMA‐based ...
Chapter 4
TABLE 1 Overview of typing technologies used for
M. tuberculosis
(380)
TABLE 2 Challenges and potential solutions in WGS‐based typing
Chapter 5
TABLE 1 Overview of the molecular‐based methods for antimicrobial resistanc...
TABLE 2 Anti‐retroviral drugs (ARV) and resistance
a
TABLE 3 Drugs used for treating herpesvirus infections, gene targets, and d...
TABLE 4 HCV directly acting antiviral drugs, gene targets, and resistance m...
TABLE 5 HBV reverse transcriptase mutations conferring resistance to NRTI
TABLE 6 Commercial molecular diagnostic methods for detection of antibacter...
Chapter 6
TABLE 1 Various sample types and their suitability for broad‐range amplific...
Chapter 8
TABLE 1 Multianalyte biomarker tests for the diagnosis of acute infections
a
Chapter 9
TABLE 1 Commercial kits for rRNA depletion
a
Chapter 11
TABLE 1 Common specimen types, transport conditions, and collection/process...
Chapter 12
TABLE 1 Commonly utilized extraction options
Chapter 14
TABLE 1 Types of internal controls
Chapter 15
TABLE 1 Recommended guidance for assessing performance characteristics of m...
TABLE 2 2x2 Table
TABLE 3 Risk acceptability matrix
a
TABLE 4 Instrument‐to‐instrument comparison study samples
a
TABLE 5 Instrument‐to‐instrument initial comparison results
a
Chapter 16
TABLE 1 Metrological definitions from ISO/IEC Guide 99 International Vocabu...
TABLE 2 List of molecular microbiology standards as of December 2024
a
Chapter 17
TABLE 1 Overview Illumina sequencers and flow cells
TABLE 2 Sequencing details for the DNBSEQ‐G99 instrument
TABLE 3 Overview of IonTorrent sequencers (39)
TABLE 4 G4 Sequencing configurations for metagenomics (41)
TABLE 5 UG 100 specifications
TABLE 6 Overview of PacBio HiFi sequencers
TABLE 7 Overview of ONT sequencing instruments (52)
Chapter 19
TABLE 1 Common read processing steps and software used for analysis
a
TABLE 2 Important quality metrics and recommended quality score thresholds ...
Chapter 20
TABLE 1 Comparison of reference mapping and
de novo
assembly
TABLE 2 Comparison of three commonly used assemblers for short‐read data
TABLE 3 Characteristics and challenges of cgMLST, wgMLST, and SNP typing
Chapter 21
TABLE 1 Representative sterile versus nonsterile sample types
a
TABLE 2 QC steps + tools
a
TABLE 3 Alignment tools
a
TABLE 4 Assembly tools
a
TABLE 5 Taxonomic assignment tools
a
TABLE 6 Sequence data repositories and taxonomies
a
TABLE 7 End‐to‐end solutions
a
Chapter 22
TABLE 1 Comparison between different assembly/binning strategies
TABLE 2 Breakdown of various methods in terms of their reference frame, the ...
Chapter 23
TABLE 1 Published validation studies for common microbiology NGS testing ap...
TABLE 2 Examples of available guidance documents
a
TABLE 3 Examples of risks and failure modes across a typical NGS workflow
a
...
TABLE 4 Source of unexpected positive and negative results
TABLE 5 Sources of samples
TABLE 6 Examples of quality risk, potential mitigations, and relevant QC me...
TABLE 7 Example sources of proficiency testing and reference materials
a
Chapter 24
TABLE 1 Demonstration of high‐confidence genotypic prediction of phenotypic...
TABLE 2 Summary of antiviral resistance loci used in clinically available N...
Chapter 25
TABLE 1 Overview of main classes of human RNA, approximate nucleotide (NT) ...
Chapter 26
TABLE 1 Comparison of phenotypic and genotypic susceptibility testing
TABLE 2
Staphylococcus aureus
and
Enterococcus
spp.: interpretation of unex...
TABLE 3
Enterobacterales
: interpretation of commonly tested beta‐lactamases...
TABLE 4
Pseudomonas aeruginosa
and
Acinetobacter baumannii
: interpretation ...
Chapter 27
TABLE 1 Weekly testing goals for novel influenza variant detection
Chapter 29
TABLE 1 Major assay design factors needing to be considered for pathogen det...
TABLE 2 Major analytical processing factors needing to be considered for seq...
TABLE 3 Major bioinformatic analysis and interpretation factors needing to b...
Chapter 30
TABLE 1 Clinical diagnostic applications for microbial NGS sequencing
TABLE 2 Key microbiome‐associated diseases by application, including inflam...
TABLE 3 Considerations for developing microbiome‐based diagnostics
Chapter 31
TABLE 1 Commercially available ultrasensitive HIV tests
a
TABLE 2 Analytic limit of detection for FDA‐cleared HSV assays used for CSF...
Chapter 32
TABLE 1 Summary of clinical performance of select syndromic panels
TABLE 2 Summary of clinical performance of select molecular tests
Chapter 33
TABLE 1 Considerations for implementing emerging diagnostics in the context...
TABLE 2 Examples of report comments that might be appropriate for antimicro...
Chapter 36
TABLE 1 Questions to ask before conducting POR
Chapter 1
FIGURE 1 Thermal nucleic acids amplification methods. (A) Conventional PCR. ...
FIGURE 2 Real‐time PCR. (A) Amplification curve. Increase in fluorescence an...
FIGURE 3 Real‐time PCR detection chemistries. (A) Intercalating dye. Binding...
FIGURE 4 Recommended unidirectional workflow. Area 1, pre‐PCR processes, cle...
Chapter 2
FIGURE 1 Identification of linkage between two targets. Example of graphs pr...
FIGURE 2 Detection of single nucleotide polymorphisms using droplet dPCR. Ex...
Chapter 3
FIGURE 1 Timeline of milestones in development and application of nucleic ac...
FIGURE 2 Timeline of milestones in the development of CRISPR‐Cas systems as ...
FIGURE 3 The principle and reaction steps of LAMP (7, 33). (Left, top) LAMP ...
FIGURE 4 The principle and reaction steps of TMA (32, 33). There are two pri...
FIGURE 5 The principle and reaction steps of HDA and SDA (1, 33). (A) HDA em...
FIGURE 6 Schematic illustration of NAATs using the CRISPR‐Cas systems for th...
Chapter 4
FIGURE 1 Critical steps in the workflow for WGS‐based typing. Three main ste...
FIGURE 2 Example of the ISO‐accredited bioinformatic pipeline “IMMense” deve...
Chapter 6
FIGURE 1 The principle and steps in 16S rRNA broad‐range amplification and s...
FIGURE 2 The structure of the fungal rRNA operon. Light grey sections V1–V9 ...
Chapter 7
FIGURE 1 Microbial composition varies across body sites while metabolic path...
Chapter 8
FIGURE 1 Each pathogen induces a specific response in the host that can be m...
FIGURE 2 Viral versus bacterial host responses. Using microarray technology,...
FIGURE 3 Modular expression in infants and young children with RSV infection...
Chapter 10
FIGURE 1 A visual representation of unidirectional workflow in different ass...
FIGURE 2 Examples of lean molecular laboratories. (A) One example of a lean ...
Chapter 15
FIGURE 1 Precision and accuracy. Precision and accuracy are often visually d...
FIGURE 2 Accuracy/agreement calculation example. A hypothetical assay is bei...
Chapter 16
FIGURE 1 (A) ISO17511 case with an international conventional calibrator tha...
FIGURE 2 The nucleic acid sample is divided into many independent partitions...
FIGURE 3 Regression analysis to evaluate commutability between two measureme...
Chapter 17
FIGURE 1 Next‐generation sequencing workflow. Illumina NGS includes four ste...
FIGURE 2 Illumina complete long‐read prep. Tagmentation is employed to fragm...
FIGURE 3 Avidity sequencing workflow and scheme. (A) Sequencing by avidity. ...
FIGURE 4 Nanoball sequencing. Illustration of the different steps of DNA nan...
FIGURE 5 Ultima genomics sequencing platform. (A) Wafer surface patterned at...
FIGURE 6 The principle of single‐molecule, real‐time (SMRT) DNA sequencing. ...
FIGURE 7 A MinION flow cell consists of 512 channels, each containing 4 nano...
Chapter 18
FIGURE 1 Most common nucleic acid separation methods. (A) Solid‐phase extrac...
FIGURE 2 Examples of gel/bioanalyzer/tape station quality control. Example o...
FIGURE 3 General library preparation steps by technology. For illustration p...
Chapter 19
FIGURE 1 FastQC output of a good quality sample. Reads were sequenced on an ...
FIGURE 2 FastQC output of a poor‐quality sample. Reads were sequenced on an ...
FIGURE 3 Read filter statistics profile. This represents a summary report of...
FIGURE 4 Read quality profile. Reads were sequenced on an Illumina NextSeq 1...
FIGURE 5 ONT accuracy versus read depth. Read depth has diminishing returns ...
FIGURE 6 Example of IGV pileup. The provided reference genome sequence is de...
FIGURE 7 Example of a Nextflow workflow.
FIGURE 8 Example of Snakemake workflow generated via AI. The figure represen...
Chapter 20
FIGURE 1 Scheme of steps to reconstruct and analyze a whole genome sequence ...
FIGURE 2 Comparison of the two approaches for genome assembly. With referenc...
FIGURE 3 Principle of allele‐based cg/wgMLST versus SNP typing. Based on ext...
FIGURE 4 Visualization of a maximum‐likelihood tree showing different annota...
Chapter 21
FIGURE 1 Targeted versus untargeted shotgun metagenomic next‐generation sequ...
FIGURE 2 High‐level summary of a typical bioinformatics workflow for clinica...
Chapter 22
FIGURE 1 Comparison of De Brujin assembly to overlap assembly.
FIGURE 2 An example of an Anvio visualization of MAGs, and their correspondi...
FIGURE 3 Rarefaction curves to quantify alpha diversity with respect to sequ...
FIGURE 4 Overview of the typical beta diversity pipeline. (A) Once a distanc...
FIGURE 5 Qualitative comparison of various distance metrics. (A) Qualitative...
FIGURE 6 Illustrative example highlighting challenges in differential relati...
FIGURE 7 An example of the output of an ILR transform applied to a 16S datas...
Chapter 24
FIGURE 1 Comparison of detection of low‐frequency mutations in CMV UL97 usin...
FIGURE 2 Genomic epidemiology input data and visualization examples. Epidemi...
FIGURE 3 The mean waiting time for a mutation as a function of alignment len...
Chapter 25
FIGURE 1 RNA‐Seq experimental steps: from extraction to library preparation ...
FIGURE 2 Visualization of host transcriptomic data. (A) A volcano plot shows...
Chapter 30
FIGURE 1 Assay complexity. This diagram illustrates the relationship between...
Chapter 32
FIGURE 1 Clinical guidance example surrounding the interpretation of the Men...
Chapter 33
FIGURE 1 Examples of teams and key stakeholders for successful implementatio...
FIGURE 2 Critical decision points for antimicrobial prescribing in serious i...
FIGURE 3
C. difficile
pre‐ and postanalytical requirements for molecular tes...
FIGURE 4 Functional teams and organizational support required for ASP and DS...
FIGURE 5 Example alert in the electronic medical record to appear when blood...
Chapter 35
FIGURE 1 PICOT Framework. The PICOT framework table illustrates the six step...
FIGURE 2 SWOT analysis. The figure illustrates a SWOT analysis divided into ...
Chapter 36
FIGURE 1 Study roadmap. Adapted from (87) Khella HWZ, Yousef GM. 2018.
Cance
...
FIGURE 2 Essential study design elements.
FIGURE 3 PICOTS template for study design with instructions for project mapp...
FIGURE 4 Clinical Lab 2.0 transition from transactional to integrative labor...
FIGURE 5 Strategies to demonstrate the value of laboratory medicine interven...
Cover Page
Table of Contents
Title Page
Copyright Page
Contributors
Preface
Author and EditorConflicts of Interest
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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edited by
RANDALL T. HAYDEN
Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee
KAREN C. CARROLL
Professor Emerita, Microbiology Division, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
JOHN P. DEKKER
Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
ALEXANDER J. McADAM
Department of Laboratory Medicine, Boston Children’s Hospital, Boston, Massachusetts
DONNA M. WOLK
Geisinger, Diagnostic Medicine Institute, Danville, PA; The Geisinger Commonwealth School of Medicine, Scranton, PA
Copyright © 2025 American Society for Microbiology. All rights reserved.
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The rights of Randall T. Hayden, Karen C. Carroll, John P. Dekker, Alexander J. McAdam, and Donna M. Wolk to be identified as the editors of the editorial material in this work have been asserted in accordance with law.
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Library of Congress Cataloging‐in‐Publication Data Applied for:Hardback ISBN: 9781683674566
Cover Design: WileyCover Image: © Viaframe/Getty Images
HEGE VANGSTEIN AAMOTSection for Research and Development,Department of Microbiology and Infection Control,Akershus University Hospital,Lørenskog, Norway;Department of Nursing, Health and Laboratory Science,Faculty of Health, Welfare and Organization,Østfold University College,Fredrikstad, Norway
MELIS N. ANAHTARDepartment of Pathology,Department of Medicine,Division of Infectious Diseases,Massachusetts General Hospital,Boston, MA;Infectious Diseases and Microbiome Program,Broad Institute of MIT and Harvard,Cambridge, MA
NEIL W. ANDERSONDepartment of Pathology,University Hospitals Health System,Case Western School of Medicine,Cleveland, OH
ROI AVRAHAMDepartment of Immunology and Regenerative Biology,Weizmann Institute of Science,Rehovot, Israel
N. ESTHER BABADYClinical Microbiology Service,Infectious Disease Service,Department of Pathology and Laboratory Medicine,Memorial Sloan Kettering Cancer CenterNew York, NY
ALLEN BATEMANCommunicable Disease Division,Wisconsin State Laboratory of Hygiene,Madison, WI
SUSAN M. BENSONSchool of Population Health,Curtin University,Perth, Australia
SARA J. BLOSSERGoldbelt Professional Services, LLCAtlanta, GA
JERRY BOONYARATANAKORNKITResearch and Development, Fort Worth Diagnostics,Fort Worth, TX
BLAKE W. BUCHANDepartment of Pathology,The Medical College of Wisconsin and Children’sWisconsin,Milwaukee, WI
ARRYN CRANEYMiraVista Diagnostics,Indianapolis, IN;Petrified Bugs, LLC,Miami, FL
AUGUSTO DULANTO CHIANGDepartment of Infectious Diseases,Vanderbilt University Medical Center,Nashville, TN
JAMES J. DUNNDepartment of Pathology,Texas Children's Hospital;Department of Pathology & Immunology,Baylor College of Medicine,Houston, TX
RUBEN DYRHOVDENDepartment of Microbiology,Haukeland University Hospital,Bergen, Norway
ADRIAN EGLIInstitute of Medical Microbiology,University of Zurich,Zurich, Switzerland
DAVID C. GASTONDepartment of Pathology, Microbiology, and Immunology,Vanderbilt University Medical Center,Nashville, TN
HEATHER L. GLASGOWDepartment of Pathology,St. Jude Children’s Research Hospital,Memphis, TN
SWATHI GOWTHAMPediatric Infectious Diseases,Geisinger Medical Center,Danville, PA
ERIN H. GRAFDepartment of Laboratory Medicine and Pathology,Mayo Clinic Arizona,Phoenix, AZ
JOCELYN HAUSERPublic Health Laboratory,DC Department of Forensic Science,Washington, DC
JULIE HIRSCHHORNLaboratory Medicine,Geisinger,Danville, PA
JIM F. HUGGETTNational Measurement Laboratory, LGC,Teddington, Middlesex;School of Biosciences & Medicine,University of Surrey,GuildfordUnited Kingdom
ROMNEY HUMPHRIESDepartment of Pathology, Microbiology and Immunology,Vanderbilt University Medical Center,Nashville TN
KATRINA KALANTARChan Zuckerberg Initiative, Science,Redwood City, CA
HALUK KAVUSLaboratory Medicine,Geisinger,Danville, PA
ØYVIND KOMMEDALDepartment of Microbiology,Haukeland University Hospital,Bergen, Norway
LYDIA A. KRASILNIKOVAInfectious Diseases and Microbiome Program,Broad Institute of MIT and Harvard,Cambridge, MA;Howard Hughes Medical Institute,Chevy Chase, MD
CHARLES R. LANGELIERDivision of Infectious Diseases,Department of Medicine,University of California San Francisco;Chan Zuckerberg Biohub,San Francisco, CA
PAIGE M.K. LARKINAmerican Society for Microbiology,Washington, DC
HUANG LINDepartment of Epidemiology and Biostatistics,University of Maryland,College Park, MD
BENJAMIN M. LIUDivision of Pathology and Laboratory Medicine,Children’s National Hospital;Department of Pediatrics,Department of Pathology,Department of Microbiology, Immunology and TropicalMedicine,The George Washington University School ofMedicine and Health Sciences;Children’s National Research Institute;The District of Columbia Center for AIDS Research,Washington, DC
S. WESLEY LONGHouston Methodist Hospital,Department of Pathology & Genomic Medicine,Houston, TX
EMILY C. LYDONDivision of Infectious Diseases,Department of Medicine,University of California San Francisco,San Francisco, CA
REBEKAH M. MARTINBD Life Sciences–Integrated Diagnostic Solutions,Becton, Dickinson and Company,Winnersh, United Kingdom
AMY J. MATHERSDepartment of Medicine and Pathology,Division of Infectious Diseases and International Health,University of Virginia,Charlottesville, VA
NINAD MEHTADivision of Clinical Microbiology,Royal University Hospital & Saskatchewan Health Authority;Department of Pathology & LaboratoryMedicine, College of Medicine,University of Saskatchewan,Saskatoon, SK, Canada
ASUNCION MEJIASDepartment of Infectious Diseases,St. Jude Children’s Research Hospital,Memphis, TN
ALEXANDER MELLMANNInstitute of Hygiene,University Hospital Muenster,University of Muenster;National Consulting Laboratory for Hemolytic UremicSyndrome (HUS);National Reference Center for Clostridioides difficile,Muenster Branch,Muenster, Germany
STEVE MILLERDelve Bio;Department of Laboratory Medicine,University of California San Francisco,San Francisco, CA
ABRAHAM G. MOLLERBacterial Pathogenesis and Antimicrobial Resistance Unit,National Institute of Allergy and Infectious Diseases,National Institutes of Health,Bethesda, MD
JACOB MORAN‐GILADSchool of Public Health,Faculty of Health Sciences,Ben Gurion University of the Negev,Beer Sheva, Israel;Department of Clinical Microbiology and InfectiousDiseases,Hadassah Medical Center,Jerusalem, Israel
JAMES T. MORTONG&A,Gutz Analytics,Boulder, CO
HEBA H. MOSTAFADepartment of Pathology,Division of Medical Microbiology,The Johns Hopkins School of Medicine,Baltimore, MD
BRIAN O’DONOVANDelve Bio,San Francisco, CA
DANIEL A. ORTIZDepartment of Microbiology,Labcorp,Royal Oak, MI
DENISE M. O’SULLIVANNational Measurement Laboratory, LGC,Teddington, Middlesex;School of Biosciences & Medicine,University of Surrey,GuildfordUnited Kingdom
BIJAL A. PARIKHDepartment of Pathology and Immunology,Washington University School of Medicine,St. Louis, MO
ELEANOR POWELLMicrobiology and Molecular Diagnostics Laboratory,Department of Pathology and Laboratory Medicine,University of Cincinnati,Cincinnati, OH
OCTAVIO RAMILODepartment of Infectious Diseases,St. Jude Children’s Research Hospital,Memphis, TN
KYLE G. RODINODepartment of Pathology and Laboratory Medicine,Perelman School of Medicine, University of Pennsylvania,Philadelphia, PA
ASHLEY M. ROONEYInstitute of Medical Microbiology,University of Zurich,Zurich, Switzerland
JOHN W. A. ROSSENLaboratory of Medical Microbiology and Infectious Diseases &Isala Academy,Isala Hospital,Zwolle, Netherlands;Department of Medical Microbiology and Infection Control,University of Groningen,University Medical Center Groningen,Groningen, The Netherlands;Department of Pathology,University of Utah School of Medicine,Salt Lake City, UT
JONATHAN SANDERSG&A,Lightweight Labware,Santa Cruz, CA
MOHAMAD R. A. SATERDay Zero Diagnostics,Watertown, MA;Department of Immunology and Infectious Diseases,Harvard T.H Chan School of Public Health,Boston, MA
NATALIE SCHERFFInstitute of Hygiene,University Hospital Muenster,University of Muenster,Muenster, Germany
ROBERT SCHLABERGIllumina, Inc.,Salt Lake City, UT
HELENA M.B. SETH‐SMITHInstitute of Medical Microbiology,University of Zurich,Zurich, Switzerland
PATRICIA J. SIMNERDepartment of Pathology,Division of Medical Microbiology,The Johns Hopkins School of Medicine,Baltimore, MD
DEREK D. N. SMITHEcotoxicology and Wildlife Health Division,Wildlife and Landscape Science Directorate,Environment and Climate Change Canada,Ottawa, Ontario, Canada
ARYEH SOLOMONDepartment of Immunology and Regenerative Biology,Weizmann Institute of Science,Rehovot, Israel
MEGHAN W. STAROLISDepartment of Molecular Infectious Disease,Quest Diagnostics,Chantilly, VA
NEERAJ K. SURANADepartment of Pediatrics,Department of Molecular Genetics and Microbiology,Department of Integrative Immunobiology,Department of Cell Biology,Duke Microbiome Center,Duke University School of Medicine,Durham, NC
CHIN YEE TANDepartment of Pediatrics,Duke University School of Medicine,Durham, NC;Duke‐NUS Medical School,Singapore;Medicine Academic Clinical Programme,Singapore Health Services,Singapore
CECILIA M. THOMPSONNorthwestern University Feinberg School of Medicine,Chicago, IL
HAUKE TÖNNIESInstitute of Hygiene,University Hospital Muenster,University of Muenster,Muenster, Germany
ANDREA D. TYLERComputational and Operational Genomics,National Microbiology Laboratory,Public Health Agency of Canada,Winnipeg, Manitoba, Canada
GABRIEL ELIAS WAGNERDiagnostic & Research Institute of Hygiene,Microbiology and Environmental Medicine,Medical University of Graz,Graz, Austria
ALEXANDRA S. WHALENational Measurement Laboratory, LGC,Teddington, Middlesex,United Kingdom
NATALIE N. WHITFIELDMedical Affairs,Inflammatix, Inc.,Sunnyvale, CA
DONNA M. WOLKGeisinger,Danville, PA;The Geisinger Commonwealth School of Medicine,Scranton, PA
QIYUN ZHUBiodesign Center for Fundamental & Applied Microbiomics,Arizona State University,Tempe, AZ
Twenty years ago, the predecessor of this book, Molecular Microbiology: Diagnostic Principles and Practice, made its first appearance. The polymerase chain reaction (PCR) was initially described less than twenty years prior to that, and molecular diagnostic tests were only just becoming widely used in clinical microbiology laboratories. The historical focus then was on building laboratory space for PCR, on how to implement such testing in a clinical setting, and on interpreting and reporting results to caregivers who were sometimes skeptical of their validity. Contamination control was a new concept, assay design was done painstakingly by a relatively few, highly trained scientists, and assays were implemented by staff familiar with largely manual methods that had only just made the transition from the research space.
The world we find ourselves in now is markedly different. PCR has become integral to clinical diagnostics and patient care as instrumentation has evolved to the point where many systems are fully automated, sometimes highly portable, and even available at the point of care. In some ways, the challenges faced in the infancy of this promising technology have been overcome. Yet, laboratories still struggle with developing and validating assays for which no commercially developed solutions are available. Contamination control is still a concern, and interpretation of results from tests whose sensitivities can far exceed those of culture or other methods is perhaps even more challenging than before.
Laboratories have access to an ever‐expanding array of instrumentation and technologies. With the introduction of high‐throughput sequencing, the landscape of the molecular microbiology laboratory is undergoing yet another major shift. Exciting applications that include routine organism identification and strain typing, assessment of molecular determinants of antimicrobial resistance, and metagenomic and transcriptomic analysis each bring new challenges in how they are best adapted to routine clinical laboratory use, when they should be ordered, which platforms and data analytic pipelines are best selected for any one application and laboratory, and how results are best interpreted and communicated. Finally, documenting the utility of molecular methods in our health care environment and selection of populations who should be tested is critically valuable to the future of molecular microbiology.
Here we provide a forward‐looking reference exploring the spectrum of new technologies and applications that promise to further revolutionize clinical microbiology and patient care. Challenges in assay development, implementation, and interpretation are discussed in depth, particularly for new, sequencing‐based modalities. Importantly, the final section of the book focuses on value‐based medicine, as clinical diagnostic stewardship takes a front seat and the added value of these important new methods to clinical care, patient outcome, and population health are examined.
This book, the Manual of Molecular Microbiology, carries forward the resounding tradition of its predecessor, while taking on the traditional moniker of other foundational texts of ASM Press that have become integral to the training and practice of clinical microbiologists worldwide. In becoming one of the hallmark “Manuals” we acknowledge that molecular microbiology has matured to mainstream clinical laboratory practice. It is no longer a separate upstart, present only in highly specialized academic centers, but it is an essential discipline with a spectrum of testing platforms that can scale and integrate into almost every imaginable practice setting, volume, complexity, and skill set. This is the world that the Manual of Molecular Microbiology now addresses. Here we hope to create a reliable source of information on both fundamentals and new technologies that managers, directors, laboratory scientists, and trainees can turn to when designing and building a laboratory, when learning about basics of the field, or when evaluating a new platform or application for deployment in an ever‐increasingly complex clinical environment.
Randall T. Hayden
Karen C. Carroll
John P. Dekker
Alexander J. McAdam
Donna M. Wolk
February 2025
Melis N. Anahtar (coauthor on chapter 24) is a cofounder, consultant, and equity holder of Day Zero Diagnostics.
Neil W. Anderson (coauthor on chapter 12) has served on scientific advisory boards for BioRad Molecular, Diasorin Molecular, and Roche.
N. Esther Babady (coauthor on chapter 1) declares research funding and advisory board service for Roche Molecular/GenMark, Bio‐Rad.
Jerry Boonyaratanakornkit (author on chapter 16) is the cofounder of Fort Worth Diagnostics.
Blake W. Buchan (coauthor on chapter 31) is a consultant to Quidel and a member of the scientific advisory board for Seegene.
Arryn Craney (author on chapter 30) is a full‐time employee of MiraVista Diagnostics, founder/owner of Petrified Bugs, and a consultant for Lighthouse Lab Services.
Julie Hirschhorn (coauthor on chapter 35) declares research funding for Abbott Molecular.
Romney Humphries (author on chapter 26) reports grants and personal fees from bioMérieux, Inc., Qiagen; grants from T2 Diagnostics, PhAST, Gradientech; and personal fees from Next Gen Diagnostics, outside the submitted work.
Katrina Kalantar (coauthor on chapters 21 and 25) is an employee of the Chan Zuckerberg Initiative and a core developer of CZ ID, a metagenomics analysis platform for researchers mentioned in the chapter.
Øyvind Kommedal (coauthor on chapter 6) is a scientific adviser, cofounder, and shareholder in Pathogenomix, Inc.
Paige M.K. Larkin (coauthor on chapter 15) has received research funding from Abbott and speaker fees from Roche.
S. Wesley Long (author on chapter 28) receives funding from BD Diagnostics.
Rebekah M. Martin (coauthor on chapter 15) is an employee of Becton, Dickinson and Company.
Ninad Mehta (coauthor on chapter 15) has received speaker fees from Hologic Inc. (Canada), is the recipient of a grant from SeeGene Open Innovation Program, and holds stocks in Hologic Inc., Pfizer, and Abbvie Inc.
Asuncion Mejias (coauthor on chapter 8) has received research grants from Janssen and Merck; fees for participation in advisory boards from Janssen, Merck, and Sanofi‐Pasteur; and fees for lectures from Sanofi‐Pasteur and Astra‐Zeneca.
Steve Miller (coauthor on chapters 21 and 29) is employed by and holds stock options at Delve Bio and holds multiple patents related to metagenomic sequencing for infectious disease.
Jacob Moran‐Gilad (coauthor on chapter 17) reports institutional grant funding from JPIAMR (EU), BSF (IL), ISF (IL), NSF (US), BGU (IL), SNF (CH) and the Government of Israel; is a scientific advisory board member at Sequentify; and is a cofounder and board member at BHT Medical.
James T. Morton (coauthor on chapter 22) is the CEO of Gutz Analytics, LLC.
Heba H. Mostafa (coauthor on chapter 5) reports grants from Bio‐Rad, DiaSorin, Qiagen, and Hologic, as well as personal fees from Seegene and BD Diagnostics.
Brian O’Donovan (coauthor on chapter 21) is an employee of Delve Bio, which is a company in the clinical metagenomics ecosystem mentioned in the chapter.
Bijal A. Parikh (coauthor on chapter 12) is on the Cepheid speaker's bureau.
Octavio Ramilo (coauthor on chapter 8) has received research grants from the Bill & Melinda Gates Foundation, Merck, and Janssen; fees for participation in advisory boards from Merck, Sanofi‐Pasteur, Pfizer, and Moderna; and fees for lectures from Pfizer, AstraZeneca, Merck, and Sanofi‐Pasteur.
Kyle G. Rodino (coauthor on chapter 13) reports consulting agreements with bioMérieux and Roche.
John W. A. Rossen (coauthor on chapter 17) has received funding or honoraria for conference attendance, advisory boards, lectures and training from ALDA, ARES‐genetics, IDbyDNA, Illumina, Molzym and Tecan. He has received research support from ZonMW (NL), EU‐cofund (H2020), JPI‐AMR (EU), Interreg (EU), the University of Groningen, EU‐Horizon, and ESCMID.
Jonathan Sanders (coauthor on chapter 22) is the CEO of Lightweight Labware.
Mohamad R. A. Sater (coauthor on chapter 24) is an employee and options holder of Day Zero Diagnostics.
Natalie Scherff (coauthor on chapter 20) is a part‐time employee of Ridom GmbH, a bioinformatics company that produces SeqSphere+, a bacterial genome analysis software mentioned in the chapter.
Robert Schlaberg (author on chapter 23) is an employee and shareholder of Illumina and is an inventor on patents licensed to or owned by Illumina.
Patricia J. Simner (coauthor on chapter 5) reports grants and personal fees from OpGen Inc., bioMérieux, Inc., Qiagen, and BD Diagnostics; grants from Affinity Biosensors and T2 Diagnostics; and personal fees from Shionogi, Inc., GeneCapture, Inoviva Specialty Therapeutics, Inc., Day Zero Diagnostics, and Next Gen Diagnostics, outside the submitted work.
Meghan W. Starolis (author on chapter 14) is a full‐time employee and stockholder at Quest Diagnostics and serves as an unpaid advisor to Roche, Bio‐Rad, and SeeGene.
Neeraj K. Surana (coauthor on chapter 7) holds stock in Pfizer.
Natalie N. Whitfield (author on chapter 36) is an employee and options holder of Inflammatix, Inc.
1 Real‐Time PCR
JOCELYN HAUSER, ELEANOR POWELL, AND N. ESTHER BABADY
2 Digital PCR
ALEXANDRA S. WHALE, DENISE M. O’SULLIVAN, AND JIM F. HUGGETT
3 Isothermal Nucleic Acid Amplification Technologies and CRISPR‐Cas‐Based Nucleic Acid Detection Strategies for Infectious Diseases Diagnostics
BENJAMIN M. LIU
4 Strain Typing (Bacterial, Viral, Fungal, and Mycobacterial)
HELENA M.B. SETH‐SMITH, ASHLEY M. ROONEY, AND ADRIAN EGLI
5 Antimicrobial Resistance Genotyping
HEBA H. MOSTAFA AND PATRICIA J. SIMNER
6 Broad‐Range Diagnostic Amplification and Detection
ØYVIND KOMMEDAL AND RUBEN DYRHOVDEN
7 Characterization of the Human Microbiome in Health and Disease
CHIN YEE TAN AND NEERAJ K. SURANA
8 Host Transcriptomics: Clinical Applications
ASUNCION MEJIAS AND OCTAVIO RAMILO
9 Organism Transcriptomics
ARYEH SOLOMON AND ROI AVRAHAM
JOCELYN HAUSER, ELEANOR POWELL, AND N. ESTHER BABADY
The polymerase chain reaction (PCR) is a well‐established method in clinical laboratories. In a series published in 2014, authors highlighted 12 transformative molecular diagnostic assays, including several infectious disease tests such as PCR for the detection of the human immunodeficiency virus (HIV) and the herpes simplex virus (HSV) (1). That series underscored the significant impact that PCR has had on medicine. While the impact of PCR on the diagnosis of diseases is undeniable, early PCR tests were cumbersome and not readily transferrable from basic research benches to high‐complexity clinical laboratories. Early works that preceded and laid the foundation for the development of PCR focused on enzymatic replication of synthetic DNA, a process coined “replication repair” (2). Further efforts followed, using only two cycles of amplifications and taking several hours to amplify a 30‐nucleotide target (3). That initial approach was labor‐intensive, low‐yield and did not achieve the potential of PCR to generate thousands of copies of DNA from a single molecule.
In 1985, Kary Mullis, a chemist working at Perkin‐Elmer Cetus Corporation in California, first described his development of PCR, which was built upon several existing concepts including dideoxy DNA sequencing as well as DNA repair and replication using DNA polymerases (4). In his approach, a PCR reaction included two oligonucleotide primers, dideoxynucleotide phosphates (ddNTPs), a DNA target, and DNA polymerase. The repeating nature of the experiment would exponentially amplify the target DNA by using prior amplicons as templates for the following amplification cycles. PCR is a cyclic process with the following steps: i) denaturation of the DNA template from double strands to single strands at 95ºC; ii) annealing of oligonucleotide primers at a lower temperature (50–65ºC); iii) primer extension by Taq polymerase. The cycle is repeated 30–45 times, resulting in detectable concentrations of DNA. Key improvements in several processes, including the use of the thermostable Taq DNA polymerase, improved oligonucleotides (oligos) chemistry, and laboratory automation (e.g., thermocyclers), resulted in the development of the modern PCR test.
The development of thermal cyclers that enabled rapid switching between different temperatures significantly reduced the time to complete a PCR reaction, which initially required manual transfer of PCR reactions between water baths kept at different temperatures. Other technical advances included the use of capillary tubes, which allowed more rapid heat exchange in the PCR reaction mix and the addition of ethidium bromide (EtBr) in the PCR reaction mix, which allowed simultaneous amplification and detection of the amplified DNA template (5–7). Several variations on this technique, including reverse transcriptase (RT)‐PCR to amplify RNA templates, were subsequently developed. Today, real‐time PCR assays are central to the diagnosis of infectious diseases and have evolved from tests performed only in high‐complexity laboratories by highly skilled medical laboratory scientists to Clinical Laboratory Improvement Amendments of 1988 (CLIA)‐waived tests that can be performed at the point‐of‐care (POC) by nonlaboratory personnel.
This chapter will review the basics of real‐time PCR, including instrumentation and detection chemistries, primer design and assay development, and quality control and quality assurance issues. Finally, clinical applications of real‐time PCR will be presented.
Real‐time PCR builds upon the basic principle of endpoint PCR. In endpoint PCR, the detection of the amplification products (amplicons) occurs once the PCR process is complete. Amplicons are mixed with an intercalating dye such as EtBr, which binds to double‐stranded DNA (dsDNA) and fluoresces when visualized on an agarose gel under ultraviolet light (Fig. 1A). In real‐time PCR, however, amplification and detection of amplicons occur simultaneously, obviating the need for postamplification manipulation of PCR products (Fig. 1B) (8).
Real‐time PCR is performed on thermocycler instruments that are designed to rapidly achieve and maintain targeted temperatures using different approaches, including heating blocks (Peltier blocks) where heat is transferred through semiconductors, or surrounding air where heat is generated either using a light bulb or heating coil (9, 10) . Thermocyclers for real‐time PCR additionally contain a fluorescence excitation source (e.g., a laser, a light‐emitting diode, or a halogen lamp) and photodetectors (e.g., charge‐coupled devices (CCDs) cameras, photodiodes, fluorimeters) to detect the fluorophore light‐emission (11). Real‐time PCR instruments come in a variety of formats to accommodate various laboratory needs. Instrumentation formats range from “sample‐to‐answer” platforms to high‐throughput instruments that are able to perform thousands of PCR assays per day.
FIGURE 1 Thermal nucleic acids amplification methods. (A) Conventional PCR. Step 1, denaturation; step 2, primers annealing step; step 3, primer extension by Taq DNA polymerase; step 4: repeat of steps 1–3 (30–40 cycles) to yield dsDNA; step 5, visualization of PCR amplicons by agarose gel electrophoresis. (B) Real‐time PCR. Same steps 1–3 as in (A); step 4: binding of DNA intercalating dye (dark green) to dsDNA; step 5, real‐time increase and detection of fluorescence (bright green). (C) Reverse transcriptase real‐time PCR. Step 1: primer annealing to RNA; step 2, reverse transcription of RNA to cDNA; step 3: degradation of the RNA strand, steps 4–7 correspond to steps 3–5 of (B). Created with BioRender.com.
In addition to the reaction components required for endpoint PCR, including primers, DNA polymerase, deoxynucleotides (dNTPs) and buffer, real‐time PCR requires the addition of fluorescent dyes. Fluorescent signals are visualized using specialized software that plot the fluorescence intensity with each cycle of amplification that produces dsDNA (Fig. 2A). The generation of fluorescence during amplification is achieved through binding of sequence‐independent or sequence‐dependent dyes or probes to the PCR products.
Intercalating fluorescent dyes are sequence‐independent dyes that bind nonspecifically to dsDNA, particularly to the DNA minor groove, and thus do not discriminate between amplicons and primer‐dimers artifacts (12). When bound to dsDNA, the dye molecules fluoresce, and that fluorescence can be measured at a specific wavelength. Although EtBr was initially used to demonstrate real‐time PCR (13), currently, the most widely used intercalating dye is thiazole green (most commonly sold as SYBR green I) and related compounds. When unbound, SYBR green emits little to no fluorescence, however, when bound to dsDNA, its fluorescence intensity increases by 1000‐fold (Fig. 2A and 3A) (14). Further analysis of amplified nucleic acids when using intercalating dyes includes melt curve analysis (Fig. 2B and 2C) to confirm specificity and detect potential contaminants or artifacts. Use of intercalating dyes such as SYBR green presents several advantages. Because the dyes bind to dsDNA in a sequence‐independent, nonspecific manner, they can be used generically in any real‐time PCR since the specificity is dependent on primer selection for the desired target. This property allows real‐time PCR assays to be cheap and easy to develop and design (15). Another advantage of intercalating dyes is their broad compatibility with typical thermocyclers on the market since the excitation and emission spectra of the dyes can be detected by the optical settings in most standard instruments (16). Utilizing intercalating dyes may require some optimization initially to determine optimal concentrations in order to prevent PCR inhibition or changes in DNA structure that could affect melting temperatures (17). Although intercalating dyes are nonspecific, some dyes preferentially bind with certain sequences; for example, SYBR green I preferentially binds to G‐C rich sequences (15). To address some of these limitations, third‐ or next‐generation intercalating dyes such as EvaGreen have been developed (18). EvaGreen has been optimized to be nonsaturating, produce low background fluorescence, and eliminate almost all inhibitory effects on the PCR reaction (16, 18). Common dyes used for real‐time PCR are listed in Table 1.
Sequence‐specific fluorescent probes are necessary to increase the specificity of real‐time PCR assays and to perform more elaborate testing and analysis, including detection of single nucleotide polymorphisms (SNPs), multiplexing to detect multiple targets, and quantitation to determine pathogen burden. These probes are oligos with a sequence complementary to a region of the target internal to the forward and reverse primers. Fluorescent probes depend on the physical process known as fluorescence (or Förster) resonance energy transfer (FRET), which relies on the transfer of energy from an excited donor fluorophore to an acceptor fluorophore. Alternatively, a fluorophore and a quencher, instead of two fluorophores, may form the pair in the process. FRET is dependent on the distance between the donor and the acceptor fluorophore as energy is transferred through intermolecular long‐range dipole‐dipole coupling and an overlap in emission and absorption spectrums of the donor and the acceptor is necessary (19).
FIGURE 2 Real‐time PCR. (A) Amplification curve. Increase in fluorescence and the resulting sigmoidal curve and cycle threshold value. (B) Melt curve analysis. After amplification is completed, the temperature is incrementally increased and the change in fluorescence of the PCR product plotted. (C) Melt curve. The Tm is the maximum peak of the first derivative of the melt curve.
FIGURE 3 Real‐time PCR detection chemistries. (A) Intercalating dye. Binding of dye (dark green) to dsDNA results in fluorescence (bright green) that is detected in real‐time (amplification curve) and further analyzed using a melt curve analysis. (B) Hydrolysis probes. Binding of probes to the complementary DNA results in fluorescence (bright green) during extension step when the fluorophore is cleaved off away from the quencher (dark grey). Fluorescence is detected in real time (amplification curve) but no melt curve is performed since the probes are hydrolyzed. (C) Molecular beacon. Binding of probes to the complementary DNA results in fluorescence (bright green) during extension step when the fluorophore is separated from the quencher (dark grey). Fluorescence is detected in real time (amplification curve) and further analyzed using melt curve analysis. (D) Hybridization probes. Binding of the donor probe (dark orange) and the acceptor probe (dark green), results in transfer of energy from the excited donor (bright orange) to the acceptor that then emits fluorescence (bright green), which can be detected in real time (amplification curve) and further analyzed using melt curve analysis. (E) MultiCode primers. Labeled primers (bright green) bind to the complementary DNA and, as amplification and extension occur, incorporation of the quencher results in a decrease in fluorescence, which can be detected in real time (amplification curve) and further analyzed using a melt curve analysis. Created with BioRender.com.
TABLE 1 Examples of commercially available DNA intercalating dyes used for real‐time PCR
Dye
Excitation peak (nm)
Emission peak (nm)
Manufacturer
SYBR Green
~498
a
~520
ThermoFisher Scientific
ResoLight
487
503
Roche Life Science
EvaGreen
500
530
Biotium
LCGreen Plus
440–470
470–520
BioFire
a Main peak, additional peaks at ~290 nm and ~380 nm.
There are several types of fluorescent probes. Among the most widely used are the hydrolysis probes, also known as TaqMan probes. These probes are labeled with a fluorescent reporter dye at the 5′ end and a quencher at the 3′ end (Fig. 3B). When the probe is intact, close proximity of the quencher prevents the reporter dye from fluorescing and producing a detectable signal. During the annealing step, the probes hybridize to the complementary target sequence. As the Taq polymerase extends and synthesizes the new complementary strand, the 5′–3′ exonuclease activity of the Taq DNA polymerase cleaves the probe, releasing the quencher and allowing the reporter to fluoresce (8, 20). The detectable fluorescence is proportional to the quantity of amplified product. With each cycle, fluorescence is expected to increase if the target sequence is present. Hydrolysis probes are widely utilized for diagnostic assays because they increase the PCR specificity, are easy to multiplex, and do not require melt curve analysis. The downside of hydrolysis probe‐based assays is the cost to synthesize custom probes for each assay in comparison to utilizing nonspecific intercalating dyes. This cost also increases with multiplexing.
In contrast to hydrolysis probes, molecular beacons do not depend on a polymerase with exonuclease activity. Molecular beacons are sequence‐specific oligos that are folded in a hairpin structure with the loop section complementary to the target sequence (Fig. 3C). The two stem regions are complementary sequences with a reporter dye and a quencher attached at the 5′ and 3′ ends, respectively. The proximity of the reporter and the quencher in the hairpin prevents fluorescence of the reporter dye. During the annealing phase of amplification, the increased temperature melts the stem of the hairpin, allowing the target sequence to hybridize with the loop. Hybridization of the molecular beacon to the complementary target causes the reporter to separate from the quencher, allowing the reporter to fluoresce (21). Molecular beacons are advantageous for real‐time PCR assays with short target sequences, for assays that require high specificity such as SNP detection, and for high‐throughput testing and multiplexing. However, molecular beacons are challenging to design and optimize because the sequence and the melting temperature of the hairpin must be precise to allow for optimal denaturation and hybridization to the target sequence.
Dual hybridization probes utilize two sequence‐specific probes: one probe contains a fluorophore at the 3′ end and acts as a donor probe, while the second probe contains a fluorophore at its 5′ end and acts as an acceptor probe (Fig. 3D). If the desired PCR target is present, the two probes hybridize to adjacent sequences on the target, about 1‐5 bases apart. Following excitation, the donor fluorophore transfers emitted energy to the acceptor fluorophore, which absorbs it, becomes excited, and in turn emits fluorescence at a longer, third wavelength. The increase in fluorescence of the acceptor is proportional to the amount of target sequences present.
MultiCode‐RTx (Luminex/Diasorin Molecular, Stillwater, MN) is an example of an alternative approach to real‐time detection of PCR amplicons. This chemistry utilizes two modified bases, 5′‐methyl‐isocytosine (Iso‐C) and isoguanine (Iso‐G), labeled with a reporter fluorophore and a quencher, respectively (Fig. 2E) (22, 23