Characterization of Plant Viruses: Methods and Protocols (Springer Protocols Handbooks)

✍ Scribed by Alangar Ishwara Bhat, Govind Pratap Rao

• Publisher: Humana
• Year: 2020
• Tongue: English
• Leaves: 539
• Category: Library

✦ Synopsis

This book provides detailed information on methodologies used in biological, serological and nucleic acid based assays for the detection, diagnosis and management of plant viruses. The content is divided into six main parts, the first of which presents techniques used in the biological characterization and transmission of viruses, while Part II covers purification and techniques concerning the physico-chemical properties of viruses. In turn, Part III focuses on in vitro expression, production of polyclonal and monoclonal antibodies; and on various serological assays including precipitin tests, ELISA, blot immunoassays, immunosorbent electron microscopy and lateral flow immunoassays. Part IV addresses the isolation of DNA and RNA from plants and nucleic acid based assays such as dot-blot, polymerase chain reaction, real-time PCR, loop-mediated isothermal amplification, rolling circle amplification, recombinase polymerase amplification, and next-generation sequencing approaches. Part V discusses cloning, sequencing, sequence analysis and the production of infectious clones, while the last part (Part VI) provides biotechnological approaches for the management of viruses. Given its scope, the book will be a valuable resource for every laboratory, student and teacher, and for everyone interested in plant virology, plant pathology, plant biology and molecular biology, offering them a practical manual on various aspects of plant viruses.

✦ Table of Contents

Foreword
Preface
About the Book
Contents
About the Authors
Abbreviations
Chapter 1: Glasshouse for Maintenance of Virus and Insect Culture
1.1 Introduction
1.2 Construction Principles of a Glasshouse
1.3 Preventing Contamination in the Glasshouse
1.4 Cleaning of Equipment
1.4.1 Glass Wares, Pestle and Mortar
1.4.2 Pipettes
1.5 Glasshouse for Insect Rearing (Maramorosch and Mahmood 2014)
1.6 Notes
References
Chapter 2: Symptoms of Virus-Infected Plants
2.1 Introduction
2.1.1 External Symptoms
2.1.1.1 Local Lesion
2.1.1.2 Systemic Symptoms
Reduction in Growth (Stunting)
Chlorosis of Plant Parts/Mosaic Patterns
Ringspots
Necrosis
Symptoms on Stem
Symptoms on Flower
Symptoms on Fruit
Symptoms in Seed/Tuber
Malformation
2.1.2 Internal Symptoms (Hull 2002)
2.1.2.1 Direct Microscopic Examination (Christie and Edwardson 1987)
Materials
Method (Direct Microscopic Examination)
2.1.2.2 Examination of Inclusion Bodies Through Staining with Trypan Blue
Materials
Method (Examination of Inclusion Bodies Through Staining with Trypan Blue)
2.1.2.3 Examination of Inclusion Bodies Through Staining with Phloxine
Materials
Method (Examination of Inclusion Bodies Through Staining with Phloxine)
2.1.2.4 Examination of Inclusion Bodies by Staining with Pyronin-Methyl Green
Materials
Method (Examination of Inclusion Bodies by Staining with Pyronin-Methyl Green)
2.1.2.5 Examination by Staining with Toluidine Blue
Materials
Method (Examination of Inclusion Bodies by Staining with Toluidine Blue)
References
Chapter 3: Isolation and Diagnosis of Virus Through Indicator Hosts
3.1 Introduction
3.2 Materials
3.3 Method
3.4 Notes
References
Chapter 4: Host Range of Viruses
4.1 Introduction
4.2 Materials
4.3 Method
4.4 Notes
References
Chapter 5: Physico-chemical Properties of Virus in Crude Sap
5.1 Introduction
5.1.1 Determination of the Dilution End Point (DEP)
5.1.2 Determination of the Thermal Inactivation Point (TIP)
5.1.3 Longevity In Vitro (LIV)
5.2 Materials
5.3 Methods
5.3.1 Determination of the Dilution End Point (DEP)
5.3.2 Determination of Thermal Inactivation Point (TEP)
5.3.3 Determination of Longevity In Vitro (LIV)
5.4 Notes
References
Chapter 6: Mechanical Sap Transmission
6.1 Introduction
6.1.1 Methods of Mechanical Transmission
6.2 Materials
6.3 Methods
6.3.1 Hand Inoculation (Kado 1972; Walkey 1991)
6.3.2 Spray Inoculation (Mandal et al. 2008)
6.4 Notes
References
Chapter 7: Transmission Through Grafting and Budding
7.1 Introduction
7.1.1 Approach Grafting
7.1.1.1 Materials
7.1.1.2 Method
7.1.2 Wedge or Top Cleft Grafting
7.1.2.1 Materials
7.1.2.2 Method
7.1.2.3 Notes (Adriance and Brison 2006; Nayudu 2008)
7.1.3 Tongue Grafting
7.1.3.1 Materials
7.1.3.2 Method
7.1.4 Leaf Patch Grafting
7.1.4.1 Materials
7.1.4.2 Method
7.1.4.3 Notes
7.1.5 Bud (Shield) Grafting
7.1.5.1 Materials
7.1.5.2 Method
7.1.5.3 Notes
7.1.6 Double Budding
7.1.7 Petiole Grafting
7.1.7.1 Materials
7.1.7.2 Method
7.1.7.3 Notes
7.1.8 Chip Bud Grafting (Wagaba et al. 2013)
7.1.8.1 Materials
7.1.8.2 Method
7.1.8.3 Notes
References
Chapter 8: Transmission Through Dodder
8.1 Introduction
8.2 Materials
8.3 Method
8.4 Notes (Hull 2002; Walkey 2012)
References
Chapter 9: Virus Transmission Through Pollen
9.1 Introduction
9.2 Materials
9.3 Method (Liu et al. 2014; Atsumi et al. 2015)
9.4 Notes (Hull 2002; Card et al. 2007; Atsumi et al. 2015)
References
Chapter 10: Transmission Through Seeds
10.1 Introduction
10.2 Materials
10.3 Method
10.4 Notes (Shepherd 1972; Hull 2014)
References
Chapter 11: Transmission of Viruses by Aphids
11.1 Introduction
11.1.1 Rearing of Virus-Free Aphids
11.2 Materials
11.3 Methods
11.3.1 Culturing of Aphids
11.3.2 Transmission of Non-persistent Viruses by Aphids
11.3.3 Transmission of Persistent Viruses
11.4 Notes (Harris and Maramorosch 1980; Dijkstra and de Jager 1998; Nayudu 2008)
References
Chapter 12: Transmission of Viruses by Leafhoppers
12.1 Introduction
12.1.1 Raising Insect Colonies (Maramorosch 1999)
12.2 Materials
12.3 Method (Transmission of Rice Tungro Spherical Virus (RTSV) by Nephotettix spp.)
12.3.1 Raising Plants and Rearing of Insects
12.3.2 Generating Virus-Infected Leafhopper Colonies and Virus Transmission
12.4 Notes (Maramorosch 1999)
References
Chapter 13: Transmission of Viruses by Whiteflies
13.1 Introduction
13.2 Materials
13.3 Method
13.3.1 Maintenance of Healthy Whitefly Culture
13.3.2 Virus Transmission
13.4 Notes (Muniyappa 1980; Nayudu 2008)
References
Chapter 14: Transmission of Viruses by Thrips
14.1 Introduction
14.2 Materials
14.3 Method
14.3.1 Rearing of Thrips (Thrips palmi)
14.3.2 Virus Transmission Studies
14.4 Notes (Ullman et al. 1997; Hull 2002)
References
Chapter 15: Transmission of Viruses Through Mealybugs
15.1 Introduction
15.2 Materials
15.3 Method
15.3.1 Rearing of Mealybug (Ferrisia virgata, Planococcus sp.) and Transmission of Piper yellow mottle virus (Genus: Badnaviru...
15.3.2 Transmission of Grapevine Leaf Roll-Associated Virus (Genus: Ampelovirus; Fam: Closteroviridae) by Planococcus ficus (T...
15.4 Notes (Roivainen 1980; Bhat et al. 2003; Tsai et al. 2010)
References
Chapter 16: Transmission of Viruses Through Beetles
16.1 Introduction
16.2 Materials
16.3 Method
16.3.1 Rearing Beetle Colony and Virus Culture
16.3.2 Beetle-Virus Transmission Assays
16.4 Notes (Smith et al. 2017)
References
Chapter 17: Transmission of Viruses Through Mites
17.1 Introduction
17.2 Materials
17.3 Method
17.3.1 Rearing of Healthy Aceria cajani
17.3.2 Transmission
17.4 Notes (Slykhuis 1955; Reddy et al. 1989; Kulkarni et al. 2002)
References
Chapter 18: Transmission of Viruses Through Fungi
18.1 Introduction
18.1.1 Isolation and Culturing of Fungal Vectors
18.1.2 Transmission Studies
18.2 Materials
18.3 Method (Transmission of Tobacco Necrosis Virus by Olpidium sp.)
18.4 Notes (Teakle 1972; Hull 2014)
References
Chapter 19: Transmission of Viruses Through Nematodes
19.1 Introduction
19.1.1 Collection, Isolation, Handling and Culturing of Nematodes
19.1.1.1 Sampling
19.1.1.2 Isolation
19.2 Materials (Isolation of Nematodes)
19.3 Method (Isolation of Nematodes by Flegg Modified Cobb´s Decanting and Sieving Technique)
19.3.1 Culturing Nematodes
19.3.2 Transmission Tests
19.4 Notes (Barker 1985; Nayudu 2008)
References
Chapter 20: Storage and Preservation of Plant Virus Cultures
20.1 Introduction
20.1.1 Desiccation of Samples
20.2 Materials
20.2.1 Desiccation of Samples
20.3 Method
20.3.1 Desiccation of Samples
20.3.2 Preservation of Virus-Infected Rice Leaves and Recovery of the Virus Through Leafhopper Vector
20.3.2.1 Materials
20.3.2.2 Method
20.3.3 Freeze Drying
20.3.3.1 Materials
20.3.3.2 Method
20.3.4 Storage at -80 C
20.3.4.1 Materials
20.3.4.2 Method
20.3.5 Preservation of Virus-Infected Plant Material in Liquid Nitrogen
20.3.5.1 Materials
20.3.5.2 Method
20.3.6 Colour Preservation of Virus-Infected Tissues
20.3.6.1 Materials
20.3.6.2 Method
20.4 Notes (Dijkstra and deJager 1998; Hull 2014)
References
Chapter 21: Purification of Plant Viruses
21.1 Introduction
21.2 Stages in Purification
21.2.1 Propagation of Virus
21.2.2 Extraction of Virus
21.2.3 Clarification of the Extract
21.2.4 Concentration of the Virus
21.2.5 Further Purification of the Virus
21.3 Materials
21.3.1 Purification of Potato Virus Y (PVY) (Genus: Potyvirus) (Moghal and Francki 1976; Bhat et al. 1997)
21.3.1.1 Materials
21.3.1.2 Method
21.3.2 Purification of Sugarcane Mosaic Virus (SCMV) (Genus: Potyvirus) (Rao et al. 1998)
21.3.2.1 Materials
21.3.2.2 Method
21.3.3 Purification of Cucumber Mosaic Virus (CMV) (Genus: Cucumovirus) (Lot et al. 1972; Bhat et al. 2004)
21.3.3.1 Materials
21.3.3.2 Method
21.3.4 Purification of Piper Yellow Mottle Virus (PYMoV) (Genus: Badnavirus) (deSilva et al. 2002)
21.3.4.1 Materials
21.3.4.2 Method
21.3.5 Purification of Cucumber Green Mottle Mosaic Virus (CGMMV) (Genus: Tobamovirus) (Mandal et al. 2008)
21.3.5.1 Materials
21.3.5.2 Method
21.3.6 Purification of Groundnut Bud Necrosis Virus (GBNV) (Genus: Tospovirus) (Reddy et al. 1992)
21.3.6.1 Materials
21.3.6.2 Method
21.3.7 Purification of Tobacco Streak Virus (Genus: Ilarvirus) (Ramiah et al. 2001)
21.3.7.1 Materials
21.3.7.2 Method
21.3.8 Purification of Potato Leaf Roll Virus (PLRV) (Genus: Luteovirus) (Rowhani and Stace-Smith 1979)
21.3.8.1 Materials
21.3.8.2 Method
21.3.9 Purification of Banana Bunchy Top Virus (BBTV) (Genus: Babuvirus) (Thomas and Dietzgen 1991)
21.3.9.1 Materials
21.3.9.2 Method
21.3.10 Purification of Cymbidium Mosaic Virus (CymMV) (Genus: Potexvirus) (Frowd and Tremaine 1977)
21.3.10.1 Materials
21.3.10.2 Method
21.3.11 Purification of Barley Yellow Dwarf Virus (BYDV) (Genus: Luteovirus) (Geske et al. 1996)
21.3.11.1 Materials
21.3.11.2 Method
21.3.12 Purification of Bean Common Mosaic Virus (BCMV) (Genus: Potyvirus) (Alberio et al. 1979)
21.3.12.1 Materials
21.3.12.2 Method
21.3.13 Purification of Lily Symptomless Virus (LSV) (Genus: Carlavirus) (Wang et al. 2007)
21.3.13.1 Materials
21.3.13.2 Method
By Sephacryl S-1000 SF GFC
By Superdex-2000 HR GFC
21.3.14 Purification of Indian Tomato Leaf Curl Virus (Genus: Begomovirus) (Muniyappa et al. 1991)
21.3.14.1 Materials
21.3.14.2 Method
21.3.15 Purification of Citrus Tristeza Virus (Genus: Closterovirus) (Bar-Joseph et al. 1985; Ozturk and Cirakoglu 2003)
21.3.15.1 Materials
21.3.15.2 Method
21.3.16 Purification of Squash Leaf Curl Virus (Genus: Curtovirus) (Cohen et al. 1983)
21.3.16.1 Materials
21.3.16.2 Method
21.3.17 Purification of Rice Tungro Bacilliform Virus (RTBV) (Genus: Tungroviurs) and Rice Turngo Spherical Virus (RTSV) (Genu...
21.3.17.1 Materials
21.3.17.2 Method
Isolation and Propagation of RTSV
Isolation and Propagation of RTBV
Purification of RTSV and RTBV
21.4 Notes (Francki 1972; Brakke 1960; Matthews 1991; Hull 2002)
References
Chapter 22: Ultraviolet Absorption Spectra of Purified Virus Preparation
22.1 Introduction
22.2 Materials
22.3 Method
22.4 Notes (Noordam 1973; Wilson and Goulding 1986)
References
Chapter 23: Electron Microscopy and Utramicrotomy
23.1 Introduction
23.2 Leaf Dip Method
23.2.1 Materials
23.2.2 Method
23.3 Electron Microscopy of Ultrathin Sections
23.3.1 Materials
23.3.2 Method
23.4 Notes (Hill 1984; Milne 1984; Roberts 1986)
References
Chapter 24: Determination of Coat Protein Molecular Weight of Viruses
24.1 Introduction
24.2 Materials
24.3 Method
24.4 Notes (Laemmli 1970; Sambrook and Russel 2001)
References
Chapter 25: Isolation of Nucleic Acid from Purified Virus and Determination of Its Nature
25.1 Introduction
25.2 Isolation of Nucleic Acid from Purified Virus Preparation
25.2.1 Materials
25.2.1.1 Method I
25.2.1.2 Method II
25.2.2 Methods
25.2.2.1 Method I
25.2.2.2 Method II
25.3 Quantification of Nucleic Acid
25.3.1 Materials
25.3.2 Method
25.4 Determination of the Nature of Viral Genome
25.4.1 Materials
25.4.2 Method
25.5 Notes (Burrin 1986; Manchester 1995)
References
Chapter 26: Agarose Gel Electrophoresis for Nucleic Acids
26.1 Introduction
26.2 Agarose Gel Electrophoresis for DNA
26.2.1 Materials
26.2.2 Method
26.3 Denaturing Formaldehyde Gel for the Analysis of RNA
26.3.1 Materials
26.3.2 Method
26.4 Notes (Simpson and Whittaker 1983; Sambrook and Russel 2001 )
References
Chapter 27: In Vitro Expression of Viral Coat Protein in Prokaryotic System and Its Purification
27.1 Introduction
27.2 Materials
27.3 Method
27.3.1 Subcloning of Virus Coat Protein (CP) in Expression Vector
27.3.2 Induction of CP Clones in Expression Cell Line
27.3.3 SDS-PAGE Analysis
27.3.4 Protein Purification
27.4 Notes (Sambrook and Russel 2001; Agarwal et al. 2009)
References
Chapter 28: Production of Polyclonal Antiserum
28.1 Introduction
28.2 Materials
28.3 Method
28.3.1 Intramuscular Injection
28.3.2 Subcutaneous Injection
28.3.3 Intravenous Injection
28.3.4 Blood (Serum) Collection
28.3.5 Processing of Antiserum
28.4 Notes (Hampton et al. 1990; Dijkstra and deJager 1998)
References
Chapter 29: Production of Monoclonal Antibody
29.1 Introduction
29.1.1 Steps Involved in Monoclonal Antibody Production
29.2 Materials
29.2.1 Special Reagents for Hybridoma Technology
29.3 Immunization
29.3.1 Materials
29.3.2 Method
29.4 Cell Fusion
29.4.1 Materials
29.4.2 Method
29.4.2.1 Preparation of Feeder Cells
29.4.2.2 Preparation of Myeloma Cells
29.4.2.3 Preparation of Spleenic B Lymphocytes for Fusion
29.4.2.4 Fusion Experiment
29.5 Screening the Hybridoma
29.5.1 Materials
29.5.2 Method
29.5.2.1 Screening Strategy
29.5.2.2 Single Cell Cloning and Sub-cloning
29.6 Production of MAbs from Hybridoma Clones
29.6.1 Laboratory Methods of Hybridoma Cultivation (Batch Culture Method: Cell Culture Flask)
29.6.2 Mouse Ascites Method
29.7 Characterization of Monoclonal Antibodies
29.7.1 Reactivity in ELISA
29.8 Notes (van Regenmortel 1986; Torrance 1995)
References
Chapter 30: Serological Tests
30.1 Introduction
30.2 Precipitin Tests
30.2.1 Tube Precipitin
30.2.1.1 Materials
30.2.1.2 Method
30.2.2 Micro-Precipitin Test
30.2.2.1 Materials
30.2.2.2 Method
30.2.3 Chloroplast Agglutination Test
30.2.3.1 Materials
30.2.3.2 Method
30.2.4 Agar Double Diffusion (Ouchterlony) Test
30.2.4.1 Materials
30.2.4.2 Method
30.3 Enzyme-Linked Immunosorbent Assay (ELISA)
30.3.1 Double-Antibody Sandwich (DAS) ELISA
30.3.1.1 Preparation of Immunoglobulins (IgG) (Avrameas 1969)
Materials
Method
30.3.1.2 Preparation of IgG-Enzyme Conjugate
Conjugation with Alkaline Phosphatase (ALP)
Materials
Method
Conjugation with Horseradish Peroxidase (HRP)
Materials
Method
Conjugation with Penicillinase
Materials
Method
30.3.1.3 Direct Antibody Sandwich ELISA (DAS-ELISA) Utilizing Alkaline Phosphatase
Materials
Method
30.3.1.4 DAS-ELISA Using Penicillinase
Materials
Method
30.3.2 F(ab´)2-ELISA
30.3.2.1 Preparation of F(ab´)2 Fragments
Materials
Method [Preparation of F(ab´)2 Fragments]
30.3.2.2 F(ab´)2-ELISA test
Materials
Method
30.3.3 Plate-Trapped or Triple-Sandwich ELISA
30.3.3.1 Materials
30.3.3.2 Method
30.3.4 Direct Antigen-Coated ELISA (DAC-ELISA)
30.3.4.1 Materials
30.3.4.2 Method
30.4 Dot Immunobinding Assay (DIBA)
30.4.1 Materials
30.4.2 Method
30.5 Tissue Blotting Immunoassay (TBIA)
30.5.1 Materials
30.5.2 Method
30.6 Electro-Blot Immunoassay (EBIA)/Western Blotting
30.6.1 Materials
30.6.2 Method
30.7 Immunosorbent Electron Microscopy (ISEM)
30.7.1 Trapping
30.7.1.1 Materials
30.7.1.2 Method
30.7.2 Decoration
30.7.2.1 Materials
30.7.2.2 Method
30.7.3 Immunogold Labelling
30.7.3.1 With Fresh Tissues
Materials
Method
30.7.3.2 With Pre-embedded Tissues
Materials
Method
30.7.3.3 With Post-embedded Tissue
Materials
Method
30.8 Immunofluorescence
30.8.1 Materials
30.8.2 Method
30.9 Lateral Flow Immunoassay Assay (LFIA)
30.9.1 Preparation and Assembly of the Strip
30.9.1.1 Test and Control Lines
30.9.1.2 Conjugate
30.9.1.3 Pad and Membrane Treatments
30.9.1.4 Membrane Materials
30.9.1.5 Material of the Sample Pad, Conjugate Release Pad and Absorbent Pad
30.9.1.6 Labels
30.9.2 Steps in Lateral Flow Immunoassay
30.9.2.1 Production of Polyclonal Antibody Against Plant Virus
30.9.2.2 Synthesis of Colloidal Gold Nanoparticles-Antibody Conjugate
Materials
Method
30.9.2.3 Assembly of LFIA Strips in Polypropylene Cassettes
Materials
Method
30.9.3 Standardization of LFIA Procedure
30.9.3.1 Materials
30.9.3.2 Method
30.10 Notes (Clark and Adams 1977; Clark et al. 1986; Hill 1984; Banttari and Goodwin 1985; Hampton et al. 1990; Koenig and Pa...
References
Chapter 31: Isolation of Total DNA from Plants
31.1 Introduction
31.2 Protocol I
31.2.1 Materials
31.2.2 Method
31.3 Protocol II
31.3.1 Materials
31.3.2 Method
31.4 Notes (Dellaporta et al. 1983; Murray and Thompson 1980; Zhang et al. 1998)
References
Chapter 32: Isolation of Total RNA from Plants
32.1 Introduction
32.2 Acid Guanidium Thiocyanate Phenol Chloroform (AGPC) Method (Chomczynski and Sacchi 1987)
32.2.1 Materials
32.2.2 Method
32.3 RNA Extraction and Column Purification
32.3.1 Materials
32.3.2 Method
32.3.2.1 Extraction of Total Nucleic Acids
32.3.2.2 Column Purification
32.4 Notes (Chomczynski and Sacchi 1987; Sambrook and Russel 2001)
References
Chapter 33: Isolation of Double-Stranded (ds) RNA from Virus-Infected Plants
33.1 Introduction
33.2 Materials
33.3 Method
33.4 Notes (Morris et al. 1983; Dodds et al. 1984)
References
Chapter 34: Dot-Blot Hybridization Technique
34.1 Introduction
34.2 Production of Radiolabelled Probe by Random Priming Method
34.2.1 Materials
34.2.2 Method
34.2.3 Notes
34.3 Production of Non-radioactive Probe by Random Primed Labelling
34.3.1 Materials
34.3.2 Method
34.3.3 Note
34.4 Production of Radiolabelled DNA Probes by Polymerase Chain Reaction
34.4.1 Materials
34.4.2 Method
34.4.3 Notes
34.5 Production of Non-radioactive DNA Probe by PCR
34.5.1 Materials
34.5.2 Method
34.6 Dot-Blot Hybridization
34.6.1 Materials
34.6.2 Method
34.6.2.1 Sample Preparation
34.6.2.2 Preparation of Membrane and Dotting Samples
34.6.2.3 Processing and Development of Membrane (When Radioactive Probe Is Used)
Pre-hybridization and Hybridization
Washing
Autoradiography
34.6.2.4 Processing and Development of Membrane (When a Non-radioactive Probe Labelled with DIG Is Used)
Materials
Pre-hybridization and Hybridization
Detection of Hybridized Bands by Colorimetric Method
Detection of Hybridized Bands by Chemiluminescence Method
34.6.3 Notes (When Radioactive Probe is Used)
34.6.4 Notes (When Non-radioactive Probe is Used)
References
Chapter 35: Polymerase Chain Reaction
35.1 Introduction
35.2 Steps in PCR
35.3 Components of PCR
35.4 Running PCR
35.5 PCR for the Amplification of DNA Viruses
35.5.1 Materials
35.5.2 Method
35.6 Reverse Transcription (RT) PCR for Detection of RNA Viruses
35.6.1 Single-Tube RT-PCR
35.6.1.1 Materials
35.6.1.2 Method
35.6.2 Two-Step RT-PCR (cDNA Synthesis Followed by PCR)
35.6.2.1 Materials
35.6.2.2 Method
35.7 Immunocapture (IC) PCR and IC-RT-PCR
35.7.1 Materials
35.7.2 Method
35.8 RT-PCR Using dsRNA Template
35.8.1 Materials
35.8.2 Method
35.9 Multiplex-PCR
35.9.1 Materials
35.9.2 Method
35.9.2.1 Isolation of Total RNA and DNA from Plants
35.9.2.2 Single-Tube Multiplex RT-PCR (mRT-PCR)
35.10 Nested PCR Assay
35.11 Notes (Hadidi et al. 1995; Candresse et al. 1998; López et al. 2009; Mumford and Seal 1997; Sambrook and Russell 2001)
References
Chapter 36: Real-Time Polymerase Chain Reaction
36.1 Introduction
36.1.1 Dyes Binding to the Double-Stranded DNA
36.1.2 Melt Curve and Detection of Non-specific Amplification
36.1.3 Fluorescent Probes Which Have Specificity for Binding to Targeted DNA
36.1.4 Quantification
36.1.5 Real-Time PCR Instrument
36.1.6 Amplification Curve and its Phases
36.1.7 Primer and Probe Design
36.1.8 Application
36.2 Performing Real-Time PCR/Real-Time RT-PCR Using SYBR-Green
36.2.1 Materials
36.2.2 Method (When RNA is Used as Template)
36.2.3 Method (When DNA is Used as Template)
36.3 Performing Real-Time RT-PCR Using TaqMan Assay
36.3.1 Materials
36.3.2 Method
36.4 Developing Standard Curve for Quantification
36.4.1 Materials
36.4.2 Method
36.5 Notes (Mackay et al. 2002; Lopez et al. 2003; Patel et al. 2016)
References
Chapter 37: DNA Microarray for Detection of Plant Viruses
37.1 Introduction
37.2 Materials
37.3 Method
37.3.1 Design and Synthesis of Microarray Slide
37.3.2 RNA Isolation
37.3.3 cDNA Synthesis and Fluorescent Labelling of cDNA
37.3.4 Hybridization and Scanning of Microarray Slide
37.4 Notes (Boonham et al. 2003; Agindotan and Perry 2007; Bystricka et al. 2005; Hadidi et al. 2004)
References
Chapter 38: Loop-Mediated Isothermal Amplification (LAMP)
38.1 Introduction
38.2 Materials
38.3 Method
38.3.1 Performing LAMP and RT-LAMP
38.3.2 Visual Detection of LAMP and RT-LAMP Products
38.4 Notes (Mori et al. 2001; Tomita et al. 2008; Zhang et al. 2014)
References
Chapter 39: Rolling Circle Amplification (RCA)
39.1 Introduction
39.2 Materials
39.3 Methods
39.4 Notes (Dean et al. 2001; Inoue-Nagata et al. 2004; Lau and Botella 2017)
References
Chapter 40: Recombinase Polymerase Amplification
40.1 Introduction
40.2 Materials
40.3 Methods (Based on Protocol of TwistDx, Cambridge, UK)
40.4 Notes
References
Chapter 41: Next-Generation Sequencing for Diagnosis of Viruses
41.1 Introduction
41.2 Chemistry Used by Different Platforms
41.3 General Workflow of NGS
41.4 Application in Plant Virology
References
Chapter 42: Cloning of PCR Product
42.1 Introduction
42.2 Isolation of Target DNA by Purification of PCR Product Through Low Melting Point (LMP) Agarose Gel
42.2.1 Materials
42.2.2 Method
42.3 Ligation of the Purified PCR Product into Vector
42.3.1 Materials
42.3.2 Method
42.3.3 Notes
42.4 Preparation of Competent E. coli Cells
42.4.1 Materials
42.4.2 Method (Mendel and Higa 1970)
42.4.3 Notes
42.5 Transformation of E. coli
42.5.1 Materials
42.5.2 Method
42.6 Selection of Transformants (Preparation of Master Plate)
42.6.1 Materials
42.6.2 Method
42.7 Screening and Identification of Positive (Recombinant) Clones
42.7.1 Rapid Disruption of Bacterial Colonies (Rapid) to Test the Size of Plasmids
42.7.1.1 Materials
42.7.1.2 Method
42.7.2 Screening Putative Recombinants by Colony PCR
42.7.2.1 Materials
42.7.2.2 Method
42.8 Recombinant Plasmid DNA Isolation and Restriction Analysis
42.8.1 Plasmid Isolation by Alkaline Lysis Method (Birnboim and Doly 1979)
42.8.1.1 Materials
42.8.1.2 Method
42.8.2 Plasmid Isolation by Modified Alkaline Lysis Method (Xiang et al. 1994)
42.8.2.1 Materials
42.8.2.2 Method
42.8.3 Confirmation of Recombinant Clones by Restriction Analysis
42.8.3.1 Materials
42.8.3.2 Method
42.8.4 Confirmation of Recombinant Clones by PCR
42.8.4.1 Materials
42.8.4.2 Method
References
Chapter 43: cDNA Synthesis and Cloning
43.1 Introduction
43.2 First and Second Strand cDNA Synthesis
43.2.1 Materials
43.2.2 Method
43.3 Ligation of DNA Fragment to the Vector DNA
43.3.1 Materials
43.3.2 Method
43.3.2.1 Linearization of Vector DNA
43.3.2.2 Dephosphorylation of Linearized Plasmid DNA
43.3.2.3 Ligation
43.4 Transformation
References
Chapter 44: DNA Sequencing
44.1 Introduction
44.2 Maxam-Gilbert Method
44.3 Chain Termination DNA Sequencing
44.4 Automated DNA Sequencing
44.5 Whole-Genome Sequencing
44.6 Next-Generation Sequencing
44.7 Notes
References
Chapter 45: Sequence Analysis and Phylogenetic Studies
45.1 Introduction
45.2 Identification of Similar Sequences
45.3 Sequence Retrieval from Databases
45.4 Detecting Open Reading Frame (ORF)
45.5 Translation of Nucleotide Sequence to Protein Sequence
45.6 Identification of Conserved Motifs and Domains
45.7 Determination of Similarity Between Sequences
45.8 Multiple Sequence Alignment
45.9 Phylogenetic Analysis
References
Chapter 46: Development of Infectious Clone of Virus
46.1 Introduction
46.1.1 Construction of Infectious Clones
46.1.2 Introduction of Infectious Clones into Plants
46.1.3 Construction of FL-cDNA Under the Control of Bacteriophage Promoter (In Vitro Transcription)
46.1.3.1 Materials
46.1.3.2 Method
46.1.4 Construction of FL-cDNA Under the Control of Cauliflower Mosaic Virus 35S Promoter (In Vivo Transcription)
46.1.4.1 Materials
46.1.4.2 Method
Development of Infectious Construct of Chilli Leaf Curl Virus
Construction of Full-Length Betasatellite Associated with Begomovirus
Agro-Inoculation of Chilli Using Begomovirus and Betasatellite Constructs
46.2 Notes
References
Chapter 47: Virus Elimination by Meristem-Tip Culture
47.1 Introduction
47.1.1 Meristem Culture Combined with Thermotherapy
47.1.2 Meristem Culture Combined with Chemotherapy
47.2 Materials
47.3 Method (Virus Elimination by Meristem-Tip Culture, Sasi and Bhat 2018)
47.4 Method (Virus Elimination by Meristem-Tip Culture Combined with Chemotherapy, Sasi and Bhat 2018)
47.5 Method (Virus Elimination by Meristem-Tip Culture Combined with Thermotherapy (Ramgareeb et al. 2010; Mishra et al. 2010)
47.6 Notes (Al-Taleb et al. 2011; Fayek et al. 2009; Mink et al. 1998; Mishra et al. 2010; Sasi and Bhat 2018)
References
Chapter 48: Virus Elimination Through Somatic Embryogenesis
48.1 Introduction
48.1.1 Virus Elimination Through Somatic Embryogenesis
48.2 Materials
48.3 Method: Virus Elimination by Somatic Embryogenesis (Sasi and Bhat 2018)
48.4 Method: Virus Elimination by Somatic Embryogenesis Combined with Chemotherapy (Sasi and Bhat 2018)
48.5 Method: Virus Elimination Through Somatic Embryogenesis in Sugarcane (Ramgareeb et al. 2010; Mishra et al. 2010)
48.6 Notes (Laux and Jurgens; 1997; Raemakers et al. 1995; Ramgareeb et al. 2010; Sasi and Bhat 2018)
References
Chapter 49: Production of Virus-Resistant Plants Through Transgenic Approaches
49.1 Introduction
49.1.1 Pathogen-Derived Genes for Plant Virus Resistance
49.1.2 Post-transcriptional Gene Silencing (RNA Silencing)
49.1.3 Viral Suppressors of RNA Silencing (VSR)
49.2 Requirements for the Development of Virus-Resistant Transgenic Plants
49.2.1 Development of Reliable Tissue Culture Regeneration System
49.2.2 Transformation Methods
49.2.2.1 Biolistic Method
49.2.2.2 Agrobacterium-Mediated Gene Transfer
49.2.3 Preparation of Gene Constructs in Binary Vectors
49.2.4 Selection and Regeneration of Transgenic Plants
49.2.5 Screening of Transgenic Plants
49.2.5.1 GUS Assay
49.2.5.2 Polymerase Chain Reaction (PCR) Assay
49.2.5.3 Southern Hybridization, Northern Hybridization and Western Blotting
49.2.6 Evaluation of Transgenic Plants
49.3 Development of Transgenic Papaya Through Coat Protein-Mediated Approach Using Biolistic
49.3.1 Preparation of Construct (Quemada et al. 1990; Fitch et al. 1990, 1992)
49.3.2 Transformation, Regeneration and Confirmation of Transgene Integration
49.3.3 Evaluation of Transgenic Papaya for Virus Resistance in the Greenhouse
49.4 Development of Transgenic Plant Through RNAi Approach Using Agrobacterium-Mediated Transformation
49.4.1 Preparation of Hairpin (HP) Construct
49.4.2 Agrobacterium-Mediated Transformation of Black Pepper (Nair and Gupta 2006; Jiby and Bhat 2011)
49.4.3 Testing of Transgenic Black Pepper for Viral Resistance in the Greenhouse
49.5 Notes
References
Chapter 50: Production of Virus-Resistant Plants Through CRISPR-Cas Technology
50.1 Introduction
50.1.1 Mechanism of CRISPR/Cas
50.1.2 Application in Plants
50.1.3 Virus Resístanse in Plants via CRISPR/Cas
50.1.3.1 DNA Virus Resístanse via CRISPR/Cas9
50.1.3.2 RNA Virus Resístanse via CRISPR/Cas9
50.2 General Outline of CRISPR/Cas9 Genome Editing in Plants
50.3 The CRISPR Cleavage Methodology Involves the Following Steps
50.4 Notes
References
Appendix: Common Conversions, Information Sources and Software of Nucleic Acids and ProteinsWeight conversion1 μg = 10-6 g1 ng...
DNA Data
Important DNA and Protein Information Sources and Software
Software for Sequence Analysis
Glossary