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Beschreibung

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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Table of Contents

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

List of Tables

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

List of Illustrations

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...

Guide

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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MANUAL OF MOLECULAR MICROBIOLOGY

FUNDAMENTALS AND APPLICATIONS

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.

Copublication by the American Society for Microbiology and John Wiley & Sons, Inc.

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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.

Limit of Liability/Disclaimer of WarrantyWhile the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy of completeness of the contents of this book and specifically disclaim any implied warranties or merchantability of fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The publisher is not providing legal, medical, or other professional services. Any reference herein to any specific commercial products, procedures, or services by trade name, trademark, manufacturer, or otherwise does not constitute or imply endorsement, recommendation, or favored status by the American Society for Microbiology (ASM). The views and opinions of the author(s) expressed in this publication do not necessarily state or reflect those of ASM, and they shall not be used to advertise or endorse any product.

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Library of Congress Cataloging‐in‐Publication Data Applied for:Hardback ISBN: 9781683674566

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Contributors

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

Preface

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

Author and Editor Conflicts of Interest

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.

section ITechnologies and Clinical Applications

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

1Real‐Time PCR

JOCELYN HAUSER, ELEANOR POWELL, AND N. ESTHER BABADY

INTRODUCTION

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

Basic Principles

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.

Detection Chemistry

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.

Fluorescent Dyes

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.

Fluorescent Probes

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.

Modified Nucleotides

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