This detailed book explores new molecular biological techniques for the detection of both antigens and antibodies of porcine diseases. Assays examined within this collection include improved methods of nucleic acid extraction, polymerase chain reaction (PCR), droplet digital PCR (ddPCR), polymerase spiral reaction (PSR), cross-priming amplification (CPA), enzyme-linked immunosorbent assay (ELISA), as well as peptide nucleic acid (PNA) based tools, aptamer-based tools, and lateral flow assays and different immune assay-based diagnostics for porcine diseases. As part of the Springer Protocols Handbooks series, the present book contains the type of in-depth knowledge on important diagnostics tools that provide a vital source of information for scientists, researchers, and students from different fields.
Authoritative and practical, Protocols for the Diagnosis of Pig Viral Diseases aims to guide researchers toward developing new technologies for the diagnosis of pig pathogens.

lgrsnf/1985.pdf

SPRINGER-VERLAG NEW YORK

United States, United States of America

Preface
Contents
About the Editors
Contributors
Chapter 1: Requirements and Preparedness for Attending a Viral Disease Outbreak in Pig Farms
1 Introduction
2 Infectious Disease Outbreak and Its Spread
3 Epidemiological ``Know-How ́ ́: An Important Pillar for Development of Surveillance System and Outbreak Preparedness
4 Investigation of Viral Disease Outbreak in Swine
4.1 Steps of Disease Outbreak Investigation and Management
5 Outbreak Investigation Steps for Infectious Diseases
5.1 Requirements for Outbreak Investigation
5.2 Descriptive Epidemiological Steps of Outbreak Investigation
5.3 Important Farm Related Risk Factors for Disease Outbreaks Are
5.4 Steps of Outbreak Investigation
5.5 Emergency Preparedness and Contingency Planning
6 Implementation of Viral Disease Control Program at Various Levels
7 Prevention and Control Measures for the Viral Disease Outbreaks in Swine Population
8 Conclusion
References
Chapter 2: Collection of Samples, Their Preservation and Transportation
1 Background
2 Introduction
3 Suitable Criteria for Collection and Transport and Preservation of Samples
4 Collection and Transport and Preservation of Samples
5 Conclusion
References
Chapter 3: Methods for Quantification of Viruses
1 Introduction
2 Infectivity Assays
2.1 Quantitative Assay (Plaque Assay)
2.2 Quantal Assay or End-Point Dilution Assay
3 Calculation of End-Point Titer
3.1 Reed-Muench Method
3.2 Improved Karber Method
3.3 Ramakrishnan Method
4 Haemadsorption Assay-Based Infectivity Assay for African Swine Fever Virus (ASFV)
5 Chemical/Physical Methods of Virus Quantitation
5.1 Direct Visualization of Virions by EM
5.2 Hemagglutination (HA) Assay
5.3 Genome Quantification by PCR
5.4 Serological Assays or Enzyme Linked Immunosorbent Assay (ELISA)
5.5 Flow Cytometry or Flow Virometry
6 Applications of Virus Quantification
6.1 Vaccine Production
6.2 Antiviral Development
6.3 Viral Therapeutics
6.4 Routine Virological Assays
7 Conclusion
References
Chapter 4: Protocols for Isolation of Genetic Materials from RNA Viruses
1 Introduction
2 Important Considerations for Purification of the RNA from Clinical Specimen
3 Lysis Buffer
3.1 Chaotropic Lysis Buffers
3.2 Guanidinium Acid-Phenol Extraction
4 Density Gradient Centrifugation
4.1 Procedure
5 Silica Technology
5.1 Step Procedure
6 Affinity Matrices
7 Purification Using Magnetic Beads
8 Separation of Double-Stranded RNA from Single-Stranded RNA
9 Filter Paper Matrix Cards
10 Storage of Purified RNA
References
Chapter 5: Multiplex PCR for Diagnosis of Porcine Diseases
1 Introduction
2 Principle of Multiplex PCR
3 Optimization of a Multiplex PCR
3.1 Designing of Primers
3.2 Reaction Component Optimization
4 Multiplex PCR Troubleshooting
5 Key Pointers for Multiplex PCR Development
6 Evaluation as a Potential Diagnostic Tool
7 Advantages of Multiplex PCR
8 Application of Multiplex PCR in Porcine Diseases
8.1 Porcine Respiratory Diseases
8.2 Porcine Reproductive Diseases
8.3 Clostridial Enteritis
8.4 Porcine Diarrhea
8.5 Porcine Parasitic Diseases
9 Conclusion
References
Chapter 6: Protocols for Isolation of Plasmid DNA
1 Introduction
2 Materials
2.1 Media (# Note 1)
2.2 Chemicals and Other
2.3 Major Equipment
2.4 Solutions and Buffers (# Note 2)
2.4.1 For Extraction and Purification of Plasmid DNA (# Notes 3 and 4)
2.4.2 For Analysis of Plasmid DNA
3 Methods of Plasmid Isolation
3.1 Classical Methods of Plasmid Purification
3.1.1 Agarose Electrophoresis Method
3.1.2 Rapid Boiling Method
3.1.3 Dye-CsC1 Gradient Method
3.1.4 Column Chromatography
3.1.5 Alkaline Lysis Method
3.2 Rapid Solid Phase Extraction Methods of Plasmid Purification
3.3 Protocols
3.3.1 Protocol 1
3.3.2 Protocol 2
3.3.3 Protocol 3
3.3.4 Protocol 4: Small Scale Extraction of Plasmid DNA by Alkaline Lysis Method
3.3.5 Protocol 5: Solid Phase Extraction Using Mini Column Kits for Plasmid Purification ()
3.4 Analysis of Plasmids
4 Notes
References
Chapter 7: Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases
1 Introduction
1.1 Principle
1.2 Lowry ́s Method
2 Materials
3 Methods
3.1 Cloning
3.1.1 Purification of Amplified PCR Product
By Using Gel Extraction Kit
By Using PCR Cleaning Kit
3.1.2 Preparation of Gene of Interest and Plasmid and Ligation
Preparation of BamHI/XhoI Cut Insert DNA (PCR Product)
Preparation of BamHI/XhoI Cut Vector DNA (PCR Product)
Gel Purification of Digested Insert DNA and Plasmid
3.1.3 Ligation of Insert and Vector
3.1.4 Preparation of Competent Cells
3.1.5 Transformation and Plating
3.1.6 Screening of Recombinant Clones
3.2 Expression in Prokaryotic Host System
3.2.1 Small Scale Production of Recombinant Protein
3.2.2 Large Scale Production of Recombinant Protein
3.3 Recombinant Protein Purification
3.4 Western Blot Analysis Using Anti-His Conjugate
3.5 Protein Estimation
3.5.1 Reagents
3.5.2 Protocol
3.5.3 Bradford Method
3.5.4 Reagents
3.5.5 Protocol
3.6 Expression of Recombinant Protein in Eukaryotic System
3.6.1 Lipofectamine-Based Transfection in Mammalian Cells
3.6.2 Cell Lysis and Protein Extraction
Chapter 8: RNA-PAGE-Based Diagnosis of Viral Diseases
1 Introduction
2 RNA-PAGE Principle
3 Sample Preparation: RNA Extraction from Clinical or Biological Samples
4 Procedure: From Setting Up PAGE Apparatus to Stained Gels Visualization
4.1 Electrophoresis in Cylindrical Gels Using Vertical Apparatus
4.2 Apparatus Setup for Gel Electrophoresis in Rectangle-Shaped Gel Slabs
4.3 Power Supply
5 Method
5.1 Preparation of Gel Plates
5.2 Preparation of Solutions Used in Gel
5.3 Assembling of the Gel
5.4 Prepare the 10% Resolving and 5% Stacking Gels
5.5 Set Up the Gel After Polymerization
5.6 Loading the Gel
5.7 Running the Gel
5.8 Disassembling the Gel
5.9 Silver Staining
References
Chapter 9: Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases
1 Introduction
2 Peptide Nucleic Acid (PNA)
3 Materials and Methods
3.1 List of Materials
3.2 Designing PNA Probes
3.3 Synthesis of PNA
3.4 Procedure for the Kaiser Test
3.4.1 Reagents for Kaiser Test
Solution A
Solution B
Solutions C
3.4.2 Kaiser Test
3.4.3 Interpretations
3.5 Characterization and Quantification of PNA Concentration
3.6 PNA Clamping PCR
3.6.1 Methods Clamping PCR
3.7 Peptide Nucleic Acid Visual Diagnostics
3.7.1 Gold Nanoparticle Synthesis
3.7.2 Visual Viral RNA Detection
3.7.3 Essentials of Visual RNA Detection Experiments
3.7.4 Viral RNA Quantification Using PNA Gold Nanoparticle Interactions
3.7.5 PNA and Other Nanoparticles for Diagnostic Applications
References
Chapter 10: Nucleic Acid Sequence-Based Amplification (NASBA) Methods and CRISPR/Cas13 System to Detect Pig Viral Diseases
1 Introduction
2 Loop-Mediated Isothermal Amplification (LAMP)
3 Nucleic Acid Sequence-Based Amplification (NASBA)
4 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
References
Chapter 11: Aptamers as Diagnostic Markers for Viral Infections of Veterinary Importance
1 Introduction
2 Selection of Aptamers
3 Application of Aptamer in Detection of Viral Diseases
4 Nucleic Acid Aptamers
5 Peptide Aptamers
6 Peptide Nucleic Acid Aptamers
7 Advantages of Aptamers
8 Future Prospects
References
Chapter 12: Antibody-Based Sensors for Pathogen Detection
1 Introduction
2 General Structure of an Antibody: An Overview
3 Biosensor/Immunosensor
4 Antibody Immobilization
4.1 Non-covalent Immobilization
4.2 Covalent Immobilization
4.3 Affinity-Based Immobilization
4.4 Recombinant Antibodies for Immobilization
5 Biosensor Types
5.1 Electrochemical Immunosensor
5.2 Optical Immunosensor
5.3 Piezoelectric Immunosensor
5.4 Thermometric Immunosensor
6 Biosensor Fabrication
6.1 Transducer
6.2 Bioreceptor
6.3 Electroactive Labels and Redox Species
6.4 Buffers
7 Methods for Biosensor Fabrication and Analyte Detection
7.1 Graphene-Based Immunosensor for Rotavirus Detection
7.2 An Interdigited Gold Micro-Electrode Impedimetric Immunosensor for Label-Free Zika-Virus (ZIKV) Detection
References
Chapter 13: Lateral Flow Assay for Diagnosis of Pig Viral Diseases
1 Introduction
2 LFA Design and Principle
3 Materials
4 Methods
4.1 Gold Nanoparticle Synthesis
4.1.1 Conjugation of mAb on GNP
4.2 Optimization of pH of the GNP and mAb Concentration for GNP-mAb Conjugation
4.2.1 Optimization of pH of GNP
4.2.2 Optimization of mAb Concentration for GNP-mAb Conjugation
4.3 Preparation of Lateral Flow Assay
4.4 LFA Test Procedure
5 Notes
References
Chapter 14: Droplet Digital PCR-Based Diagnosis for Porcine Viral Diseases
1 Introduction
2 ddPCR Experimental Workflow
2.1 Preparation of Reaction Mixture
2.2 Droplet Generation
2.3 PCR Amplification
2.4 Droplet Reading and Data Analysis
3 Notes
4 ddPCR for Porcine Viruses
5 Conclusion
References
Chapter 15: Protocols for Immunofluorescence Techniques
1 Introduction
1.1 Direct Immunofluorescence
1.2 Indirect Immunofluorescence
1.3 Multicolor Immunofluorescence
2 Materials
3 Method
3.1 Sample Preparation
3.1.1 In Case of Adherent Culture
3.1.2 Suspension Cell Culture
3.1.3 Tissue Samples
3.2 Fixation of Cells
3.2.1 Cell Fixation for Membrane-Associated Antigens
3.2.2 Cell Fixation for Intracellular Antigens
3.3 Permeabilization of Cell Membrane
3.4 Blocking of Non-specific Sites for Antibody Binding
3.5 Incubation with Primary Antibody
3.6 Incubation with Secondary Antibody
3.7 Counterstaining and Mounting
3.8 Imaging
3.9 Post-imaging Analysis and Issues Encountered in IF Staining
3.9.1 Specificity Validation of Antibodies
3.9.2 Photobleaching
3.9.3 Autofluorescence
3.9.4 High Background Fluorescence
3.9.5 Fluorophore Overlap
4 Notes
5 Conclusion
References
Chapter 16: Polymerase Spiral Reaction (PSR) for the Diagnosis of Porcine Viral Diseases
1 Introduction
2 Materials Required for PSR
3 Methods
3.1 Primer Designing
3.2 Procedure
3.3 Determination of Specificity of PSR
4 Notes
References
Chapter 17: Recombinase Polymerase Amplification-Based Diagnostics of Porcine Viral Diseases
1 Introduction
2 RPA Mechanism
3 RPA Operating Parameters
3.1 Primers
3.2 Template
3.3 Temperature
3.4 Incubation Time
3.5 Inhibitors
3.6 Specificity and Sensitivity
3.7 Multiplexing
4 Detection of Amplicons
5 RPAs for Porcine Viruses
6 Conclusion
References
Chapter 18: Cell Culture System for Porcine Virus Isolation and Propagation
1 Introduction
2 Types of Cell Culture System
2.1 Primary and Secondary Cell Culture
2.2 Cell Lines
3 Cell Culture System for Porcine DNA Viruses
3.1 African Swine Fever Virus (ASFV)
3.2 Porcine Circovirus
3.3 Herpesviruses of Swine
3.4 Porcine Parvovirus
3.5 Porcine Adenovirus (PAdV)
3.6 Swinepox Virus
4 Cell Culture System for Porcine RNA Viruses
4.1 Classical Swine Fever Virus
4.2 Foot and Mouth Disease Virus
4.3 Porcine Rotavirus
4.4 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
4.5 Porcine Coronaviruses
4.5.1 Transmissible Gastroenteritis Virus (TGEV)
4.5.2 Porcine Epidemic Diarrhea Virus (PEDV)
4.6 Swine Influenza Virus
4.7 Japanese Encephalitis Virus
4.8 Other Vesicular Disease Viruses
4.8.1 Vesicular Stomatitis Virus (VSV)
4.8.2 Swine Vesicular Disease Virus (SVDV)
4.8.3 Swine Vesicular Exanthema Virus
References
Chapter 19: An Overview of Mouse Monoclonal Antibody Production
1 Monoclonal Antibody (mAb)
2 How Hybridomas Are Produced?
2.1 Antigen/Immunization Materials
2.2 Production of Antigen
2.3 Mice for mAb Production
2.4 Immunization of Mice
2.5 Revival of Myeloma Cells and Feeder Cells Preparation
2.6 Fusion
2.7 Single-Cell Cloning
2.8 Bulk Production
2.9 Purification
3 Characterization of mAb
4 Application of mAbs in Diagnosis
4.1 Detection of Antigen and Antibody
4.2 Antigenic Profiling of Viruses
5 Conclusion
References
Chapter 20: Nucleic Acid Hybridization Techniques for Viral Disease Diagnosis: A Detailed Perspective
1 Introduction
2 Sample Collection
3 Sample Preparation
3.1 Stool Samples
3.1.1 RNA Isolation
3.1.2 DNA Isolation
3.2 Nasal Swabs
3.2.1 RNA Isolation
3.2.2 DNA Isolation
4 Probes
4.1 Preparation and General Considerations
4.2 Probe Labeling
4.2.1 Radioactive Labeled Probes
4.2.2 Non-radioactive Labeled Probes
5 Optimizing Conditions for Hybridization
5.1 Temperature
5.2 Probe Concentration
5.3 Hybridization Time
5.4 Buffer Components
6 Nucleic Acid Hybridization Protocol
6.1 Southern Blotting Using Radioactive Labeled Probe
6.1.1 Protocol
6.2 Northern Blotting Using Radioactive Labeled Probes
6.2.1 Protocol
6.3 Spot Hybridization
6.3.1 Protocol
7 Advantages and Limitations of Nucleic Acid Hybridization Methods
7.1 Advantages
7.2 Limitations
8 Applications of Nucleic Acid Hybridization in Viral Disease Diagnosis
8.1 Applications in Rapid Viral Disease Diagnosis
8.2 Applications in Histopathology
8.3 Applications in Viral Disease Epidemiology
9 Conclusion
References
Chapter 21: Ligase Detection Reaction-Fluorescent Microsphere Assay
1 Introduction
2 Principles of LDR
3 Principles of LDR-Fluorescent Microsphere Assay (LDR-FMA)
3.1 Detection
3.2 Signal Amplification
3.3 Hybridization and Readout
4 Materials
5 Methods
References
Chapter 22: ELISA as a Diagnostic Weapon
1 Introduction
1.1 ELISA as a Diagnostic Weapon for FMD
1.2 Timeline of Invention of Different Types of ELISA
1.3 Direct ELISA
1.4 Indirect ELISA
1.5 Sandwich ELISA
1.6 Competitive/Inhibition ELISA
2 Materials
2.1 Solid-Phase Competitive ELISA for the Diagnosis of FMD
2.2 Liquid Phase Blocking ELISA for Diagnosis of FMD
2.3 Indirect ELISA to Detect Non-structural Protein (NSP ELISA)
3 Methods
3.1 Solid-Phase Competitive ELISA for the Diagnosis of FMD
3.2 Liquid Phase Blocking ELISA (LPBE) for Diagnosis of FMD
3.3 Indirect ELISA to Detect Non-structural Protein (NSP ELISA)
4 Notes
References
Chapter 23: SDS-PAGE and Western Blotting: Basic Principles and Protocol
1 Introduction
2 Sample Preparation
3 Electrophoresis
4 SDS-PAGE
5 Native PAGE
6 Protocol for SDS-PAGE
6.1 Materials Required
6.2 Procedure
6.2.1 Casting of Gel
6.2.2 Details of Gel Preparation
6.2.3 Stacking Gel
6.2.4 Sample Preparation and Loading
6.2.5 Staining of Polyacrylamide Gel with Coomassie Brilliant Blue
6.2.6 Electrotransfer
6.2.7 Blocking
6.2.8 Probing
6.2.9 Detection of Bound Antibody
6.2.10 Stripping and Re-probing
7 Protocol for Western Blot
7.1 Reagents and Materials Required for Blotting
7.2 Procedure
7.2.1 Protein Blotting (Semidry Method) and Developing
7.2.2 Precautions
8 Applications of PAGE and Western Blotting in Virology
9 Limitations of Western Blot
10 Troubleshooting
11 Conclusion
References
Chapter 24: Immune Assays as Diagnostic for Pig Viral Diseases
1 Introduction
2 Viral Diseases of Pigs and Their Public Health Importance
3 Diagnostic Methods for Viral Diseases: Needs and Challenges
4 Recent Methods in the Diagnosis of Viral Infections
4.1 Immunoassay-based Tests
4.1.1 Radio-immunoassay (RIA)
4.1.2 Enzyme Immunoassays
4.1.3 Precipitation and Particle Immunoassays
4.1.4 Multiplex Immunoassays
4.2 Amplification-based Assays
4.3 Next-Generation Sequencing
4.4 Mass Spectrometry
5 Immune Assays in Swine Viral Diseases
6 Immunoassays and Immunochemistry
7 Swine Viral Diseases and Major Diagnostics Immunoassays
7.1 African Swine Fever
7.2 Aujeszky ́s Disease
7.3 Blue Eye Disease
7.4 Bovine Viral Diarrhea Virus
7.5 Classical Swine Fever
7.6 Delta Coronavirus
7.7 Ebola Reston Virus
7.8 Encephalomyocarditis
7.9 Enteroviruses
7.10 Foot-and-Mouth Disease
7.11 Hepatitis E Virus
7.12 Influenza
7.13 Japanese B Encephalitis
7.14 Nipah Virus Disease
7.15 PRRS
7.16 Parainfluenza
7.17 Porcine Circovirosis
7.18 Porcine Cytomegalovirus
7.19 Porcine Epidemic Diarrhea
7.20 Porcine Parvovirus Infection
7.21 Rotavirus Infection
7.22 Senecavirus A
7.23 Teschen Disease
7.24 Transmissible Gastroenteritis
7.25 Vesicular Exanthema
7.26 Vesicular Stomatitis
8 Conclusion
References
Chapter 25: Production of Virus-like Particles Using the Baculovirus Expression System and Their Application in Vaccines and V...
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture and Media Requirement
3.1.1 Bacterial Strain and Insect Cell Lines
3.1.2 Culture Conditions and Stock Preparation
3.2 Generation of Recombinant Baculovirus Expressing Gene of Interest
3.3 Detection of Recombinant Baculoviral Titer by Plaque Assay
3.3.1 Baculoviral Titer by Plaque Assay
3.3.2 Titration by Sf9 ET Cell
3.4 Purification of Empty Capsids from Insect Cell
3.5 Analysis of Empty Capsids
3.5.1 Analysis of Expression of Capsid Protein by Coomassie Staining
3.5.2 Analysis of Expression of Capsid Protein by Western Blot
3.5.3 Analysis of Capsids Protein Assembly by Transmission Electron Microscopy (TEM)
3.5.4 Detection of Antigenicity by Immunogold Staining
3.6 Detection of Yield of Empty Capsid by ELISA
3.7 Use of Empty Capsid to Detect Serum Antibody by ELISA
4 Notes
References
Chapter 26: Good Laboratory Practices and Biosafety Containments in a Virology Laboratory
1 Introduction
2 History of Lab-Escaped Pathogens
2.1 Smallpox Virus in 1966-1978
2.2 Human Influenza H1N1 in 1977
2.3 Venezuelan Equine Encephalitis (VEE) in 1995
2.4 SARS (Severe Acute Respiratory Syndrome) Virus in 2002-2003
3 Good Laboratory Practices
4 Biosafety Requirements While Working with Infectious Agents
5 Risk Groups, Biosafety Levels, and Physical Containment Levels
6 Working Safely in a Virology Laboratory
7 Conclusion
References
Index

2024-04-22