Preface
Contents
Contributors
Part I: Reviews
Chapter 1: Multiplex Technologies in COVID-19 Research, Diagnostics, and Prognostics: Battling the Pandemic
1 Introduction
2 SARS-CoV-2
3 Multiplex Molecular Analysis of SARS-CoV-2
3.1 Quantitative Polymerase Chain Reaction (qPCR)
3.2 MS Proteomics
3.2.1 MS-Based Techniques to Identify Diagnostic/Prognostic Biomarkers
3.2.2 MS-Based Proteomic Approaches to Monitor the Development of New COVID-19 Treatments
3.3 Multiplex Immunoassay
3.3.1 Multiplex Immunoassay for Diagnosis of COVID-19
3.4 Metabolomics
3.4.1 Metabolomic Studies of COVID-19
3.4.2 Metabolomic Identification of COVID-19 Treatment-Related Biomarkers
3.5 Other Approaches
4 Summary and Future Perspectives
References
Chapter 2: Multivalent Vaccine Strategies in Battling the Emergence of COVID-19 Variants
1 Introduction
2 Traditional Vaccine Strategies
2.1 Non-replicating (Inactivated) Viral Vector
2.2 RNA-Based
2.3 Inactivated Virus
3 The Problem with the SARS-COV-2 Variants
3.1 Alpha Variant (B.1.1.7)
3.2 Beta Variant (B.1.351)
3.3 Gamma Variant (P.1)
3.4 Delta Variant (B.1.617)
4 Multivalent Approaches to SARS-CoV-2 Vaccine Production
4.1 Constructs Using Ferritin-Based Nanoparticles
4.2 Other Multivalent Vaccine Approaches
4.3 Multivalent Vaccine Constructs Aimed at Reducing Allergenic and Toxic Side Effects
5 Conclusions and Future Perspectives
References
Chapter 3: Challenges of Multiplex Assays for COVID-19 Research: A Machine Learning Perspective
1 Introduction
2 Types of Machine Learning
3 SARS-CoV-2
4 Models Using Multiplex Profiling Data
5 Models Incorporating Laboratory Data
6 Models Incorporating Clinical and Imaging Data
7 Conclusions and Future Perspectives
References
Part II: Protocols
Chapter 4: Multiplex Quantitative Polymerase Chain Reaction Diagnostic Test for SARS-CoV-2 and Influenza A/B Viruses
1 Introduction
2 Materials
2.1 Sample Collection
2.2 qPCR
3 Methods
3.1 Nasopharyngeal Sample Collection
3.2 Saliva Sample Collection
3.3 qPCR (See Note 12)
4 Notes
References
Chapter 5: Multiplex Quantitative Polymerase Chain Reaction Test to Identify SARS-CoV-2 Variants
1 Introduction
2 Materials
2.1 Sample Collection
2.2 qPCR
3 Methods
3.1 Sample Collection
3.2 qPCR (See Note 10)
4 Notes
References
Chapter 6: NIRVANA for Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and Co-infections of Multiple Respirator...
1 Introduction
2 Materials
2.1 RNA Extraction
2.2 Reverse Transcription
2.3 RPA and DNA Purification
2.4 Agarose Gel Electrophoresis
2.5 Library Preparation and Sequencing
3 Methods
3.1 RNA Extraction
3.2 Reverse Transcription
3.3 Multiplex RPA
3.4 DNA Purification
3.5 Library Preparation and Sequencing
3.6 Real-Time Analysis
4 Notes
References
Chapter 7: Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2
1 Introduction
2 Materials
2.1 Human Clinical Samples and RNA Extraction
2.2 Real-Time One-Step RT-PCR
3 Methods
3.1 Extraction of Viral RNA from Human Clinical Samples
3.2 RT-PCR
4 Notes
References
Chapter 8: Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations
1 Introduction
1.1 Background
1.2 Special Considerations
2 Materials
2.1 Equipment
2.2 Reagents and Consumables
2.3 Specimens (See Note 2)
3 Methods
3.1 Culture Specimen Preparation (See Note 8)
3.2 SARS-CoV-2 Specimen Preparation (See Note 12)
3.3 SARS-CoV-2 Panel Preparation (See Note 14)
3.4 Assay Testing (See Note 18)
3.5 Result Analysis (See Note 20)
3.6 Further Considerations and Adaptations (See Note 33)
4 Notes
References
Chapter 9: A Rapid User-Friendly Lab-on-a-Chip Microarray Platform for Detection of SARS-CoV-2 Variants
1 Introduction
1.1 Background
1.2 Aims
1.3 Special Note
2 Materials
2.1 Sample Collection from COVID-19 Patients
2.2 PCR Labeling of SARS-CoV-2 Nucleic Acids
2.3 DNA Extraction from MRSA
2.4 PCR Amplification and Labeling
2.5 Agarose Gel Electrophoresis
2.6 PCR Purification
2.7 DNA Digestion
2.8 DNA Microarray Fabrication
2.9 Microarray Hybridization, Washing, and Scanning
2.10 Automated Hybridization, Washing, and Readout (LOC Platform)
3 Methods
3.1 Nasopharyngeal Sample Collection for SARS-CoV-2 Analysis
3.2 qPCR (See Note 15)
3.3 MRSA Genomic DNA Extraction
3.4 Standard PCR Amplification (See Note 22)
3.5 Labeling PCR Amplification
3.6 Purification of PCR Products (See Note 24)
3.7 DNA Fragmentation (See Note 25)
3.8 Microarray Fabrication
3.9 Microarray Hybridization, Washing, and Image Acquisition
3.10 Automated Hybridization, Washing, and Readout (Fraunhofer LOC Platform)
3.11 Data Analysis (See Note 33)
4 Notes
References
Chapter 10: SARS-CoV-2 Host Immunogenetic Biomarkers
1 Introduction
1.1 Background
1.2 Special Considerations
2 Materials
2.1 Equipment
2.2 Reagents and Consumables
2.3 Specimen Requirements and Handling
3 Methods
3.1 DNA and RNA Extraction (See Note 7)
3.2 SSOP PCR Using Luminex 100/200 System
3.3 HLA Typing Using NGS (See Note 47)
3.4 BCR and TCR Sequencing Using Illumina MiSeq
4 Notes
References
Chapter 11: FnCas9 Editor Linked Uniform Detection Assay for COVID-19
1 Introduction
2 Materials
2.1 Sample Collection
2.2 RNA Extraction
2.3 Single Step RT and PCR
2.4 Lateral Flow Assay
3 Methods
3.1 Extraction of RNA from Respiratory Samples (See Note 6)
3.2 Setting Up Single Step Reverse Transcription (RT)-PCR (See Note 7)
3.3 Preparation of CRISPR-Cas9 Chimeric crRNAs/Guide RNAs (See Note 8)
3.4 Preparation of RNP Mix for FELUDA (See Note 11)
3.5 Dipstick Assay Reference
3.6 TOPSE (See Note 13)
4 Notes
References
Chapter 12: Mass Spectrometry Multiplexed Detection of SARS-CoV-2
1 Introduction
2 Materials
2.1 Production of SARS-CoV-2 Recombinant Nucleoprotein
2.2 Sample Processing
2.3 LC-MS/MS Analysis
3 Methods
3.1 Production of Labeled and Unlabeled Recombinant SARS-CoV-2 Nucleoprotein
3.2 Preparation of Standard Solutions, Calibration Curves, and Quality Control Material
3.3 Sample Processing (See Note 7)
3.4 LC-MS/MS Analysis
3.5 Data Processing
4 Notes
References
Chapter 13: Identification of Circulating Biomarkers of COVID-19 Using MALDI-TOF Mass Spectrometry
1 Introduction
2 Materials (See Fig. 1) (See Note 1)
3 Methods
3.1 StageTip Assembly
3.2 Sample Preparation
3.3 MALDI-TOF Acquisition
4 Notes
References
Chapter 14: Antibody-Based Affinity Capture Combined with LC-MS Analysis for Identification of COVID-19 Disease Serum Biomarke...
1 Introduction
2 Materials
2.1 Clinical Samples (See Note 1)
2.2 Affinity Purification/Depletion
2.3 Trypsin Digestion
2.4 Liquid Chromatography-Mass Spectrometry (LC-MS)
3 Methods
3.1 Samples
3.2 Affinity Purification
3.3 Trypsin Digestion
3.4 LC-MS
3.5 Data Analysis
4 Notes
References
Chapter 15: Liquid Chromatography-Mass Spectrometry Analysis of Peripheral Blood Mononuclear Cells from SARS-CoV-2 Infected Pa...
1 Introduction
2 Materials
2.1 Sample Collection
2.2 PBMC Preparation and Fractionation
2.3 LC-MS
3 Methods
3.1 Venipuncture (See Note 1)
3.2 PBMC Preparation
3.3 Cell Culture
3.4 PBMC Fractionation (See Fig. 2)
3.5 LC-MS
4 Notes
References
Chapter 16: Assay of Fatty Acids and Their Role in the Prevention and Treatment of COVID-19
1 Introduction
2 Materials
2.1 Samples and Reagents
2.2 Instruments and Supplies
3 Methods
3.1 Fatty Acid Extraction, Hydrolysis, and Derivatization
3.2 Standard Curve Preparation
3.3 Data Analysis
4 Notes
References
Chapter 17: Lab-on-a-Chip Immunoassay for Prediction of Severe COVID-19 Disease
1 Introduction
2 Materials
2.1 Participants and Samples (See Note 1)
2.2 Microarray
2.3 Immunoassay
2.4 Equipment
3 Methods
3.1 Samples
3.2 Microarray Fabrication
3.3 Immunoassay (See Note 12)
3.4 Automated Immunoassay Procedure with the Fraunhofer LOC Platform
3.5 Data Analysis
4 Notes
References
Chapter 18: Multiplex Immunoassay for Prediction of Disease Severity Associated with the Cytokine Storm in COVID-19 Cases
1 Introduction
2 Materials
2.1 Participants and Samples (See Note 1)
2.2 Microbead Conjugation
2.3 Detection Antibodies
2.4 Multiplex Development
3 Methods
3.1 Sample Collection
3.2 Sample Processing
3.3 Antibody-Microbead Conjugation (See Fig. 3)
3.4 Biotinylation of Detection Antibodies (See Fig. 4)
3.5 Assay (See Fig. 5)
3.6 Data Analysis
4 Notes
References
Chapter 19: Detection of IgG Antibodies to SARS-CoV-2 and Neutralizing Capabilities Using the Luminex xMAP SARS-CoV-2 Multi-An...
1 Introduction
2 Materials
2.1 xMAP SARS-CoV-2 Multi-Antigen IgG Assay Kit Components
2.2 Samples and Reagents
2.3 Equipment
3 Methods
3.1 Assay Procedure (See Note 3)
3.2 System Software Setup (See Note 8)
3.3 Instrument Preparation and Data Acquisition (See Note 11)
3.4 Data Analysis
3.5 xMAP SARS-CoV-2 Multi-Antigen IgG Assay (See Notes 1 and 18)
3.6 Dried Blood Spot (DBS) Samples (See Note 20)
3.7 Isotyping (See Note 22)
3.8 Neutralizing Antibody Detection
4 Notes
References
Chapter 20: Multiplex Testing of the Effect of Statins on Disease Severity Risk in COVID-19 Cases
1 Introduction
2 Materials
2.1 Participants and Samples (See Note 1)
2.2 Preparation of Capture Antibody-Bead Conjugates
2.3 Preparation of Detection Antibodies
2.4 Multiplex Development
3 Methods
3.1 Sample Collection
3.2 Sample Processing
3.3 Antibody-Microbead Conjugation (See Note 10)
3.4 Biotinylation of Detection Antibodies
3.5 Assay
3.6 Data Analysis
4 Notes
References
Chapter 21: Evaluating the Effects of Curcumin on the Cytokine Storm in COVID-19 Using a Chip-Based Multiplex Analysis
1 Introduction
2 Materials
2.1 Participants and Samples (See Note 1)
2.2 Multiplex Development
3 Methods
3.1 Preparation of Working Reagents
3.2 Sample Collection
3.3 Sample Processing
3.4 Assay
3.5 Imaging (See Note 15)
3.6 Data Analysis
4 Notes
References
Chapter 22: COVID-19 Detection Using the NHS Lateral Flow Test Kit
1 Introduction
2 Materials (See Note 1)
3 Methods
3.1 Preparation for the Home Test (See Note 4)
3.2 Taking the Throat/Nasal Sample (See Note 5)
3.3 Processing the Sample
3.4 Testing the Sample and Reading the Result
4 Notes
References
Chapter 23: Evaluation Protocol for SARS-CoV-2 Serological Assays
1 Introduction
1.1 Background
1.2 Ease of Use
1.3 Special Considerations
2 Materials
2.1 Equipment
2.2 Reagents and Consumables
2.3 Sample Selection
3 Methods (See Note 5)
3.1 Sample Collection and Processing
3.2 Calibration and Quality Control
3.3 Automated Serology Testing (See Note 15)
3.4 Lateral Flow Immunoassays/Point of Care Immunoassays (See Note 21)
3.5 Precision
3.6 Limit of Detection
3.7 Analysis
3.8 Statistical Analysis
4 Notes
References
Chapter 24: Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs)
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods (See Note 6)
3.1 PBMC Isolation and Cryopreservation
3.2 XFp Cell Mito Stress Test Preparation
3.3 XFp Cell Mito Stress Test
4 Notes
References
Chapter 25: Multiplex Testing of Oxidative-Reductive Pathway in Patients with COVID-19
1 Introduction
2 Materials
2.1 Participants, Samples, and Reagents
2.2 TAC Analysis
2.3 Equipment
3 Methods
3.1 Treatment
3.2 Blood Sampling and Laboratory Analyses
3.3 TAC Assay
3.4 Statistics
4 Notes
References
Chapter 26: Point-of-Care Device for Assessment of Blood Coagulation Status in COVID-19 Patients
1 Introduction
2 Materials
3 Methods
3.1 Determination of INR
3.2 Optional: Use of the Coagu-App
4 Notes
References
Chapter 27: COVID-19 and the Assessment of Coenzyme Q10
1 Introduction
2 Materials
2.1 Tissues and Apparatus (See Note 1)
2.2 Extraction of CoQ10
2.3 HPLC Analysis
2.4 Total Protein Determination
2.5 Citrate Synthase (CS) Assay
3 Methods
3.1 Synthesis of the Internal Standard
3.2 Preparation of Tissue Sample
3.3 CoQ10 Extraction
3.4 HPLC Analysis (See Note 9)
3.5 Total Protein Determination (See Note 13)
3.6 Citrate Synthase Assay (See Note 15)
4 Notes
References
Chapter 28: Isolation and Cell Culture of Human Nasopharyngeal Cells: A Model for Testing Immune Response and Antiviral Treatm...
1 Introduction
2 Materials
2.1 Nasal Wash Collection
2.2 Nasal Cell Isolation (See Note 1)
2.3 Sample Characterization
3 Methods
3.1 Procedure for Nasal Wash Collection from Patients with COVID-19 (See Fig. 1) (See Note 6)
3.2 Nasal Cell Isolation and Treatment (See Note 7)
3.3 Cell Characterization
3.4 qPCR
4 Notes
References
Chapter 29: Machine Learning Approaches to Analyze MALDI-TOF Mass Spectrometry Protein Profiles
1 Introduction
2 Materials
2.1 Dataset
2.2 R Software and Packages
3 Methods
3.1 Data Preprocessing and Machine Learning Without Feature Selection (See Note 3)
3.2 Data Preprocessing and Machine Learning with Feature Selection
4 Notes
References
Chapter 30: A Deep Learning Approach to Identify Chest Computed Tomography Features for Prediction of SARS-CoV-2 Infection Out...
1 Introduction
2 Materials
3 Methods
3.1 Diagnosis and Chest CT Scan
3.2 Statistical Comparison Between Groups
3.3 Deep Learning Analysis and Classification
4 Notes
References
Part III: Future Perspectives
Chapter 31: Genomic Surveillance for Monitoring Variants of Concern: SARS-CoV-2 Delta, Omicron, and Beyond
1 Introduction
2 How Do SARS-CoV-2 Variants Arise?
3 Surveillance Techniques
4 Omicron SARS-CoV-2 Variant
5 Conclusions and Future Perspectives
References
Index