This book provides protocols for nucleic acid-based methods currently applied to plant pathogen detection and identification, showing that great strides have been made in molecular diagnostics since those first tentative steps back in the late 1980s/early 1990s. It takes the practitioner through the full range of molecular diagnostic and detection methods and, as these generic techniques are appropriate for use on any target with minimal modification, also provides a useful resource for students of plant pathology and plant pathologists. Beginning with the background and future directions of the science, it then addresses DNA barcoding, microarrays, polymerase chain reactions (PCR), quality assurance and more, forming a complete reference on the subject.

• Molecular Methods in Plant Disease Diagnostics
• Copyright
• Contents
• Editors and Contributors
• Foreword
• 1: Introduction: advances in plant diagnostics – historical perspectives and future directions
  • 1.1 Progress in recent years
  • 1.2 The rise and rise of PCR
  • 1.3 Quality matters
  • 1.4 The move from laboratory to field
  • 1.5 Identifying the unknown
  • 1.6 Future challenges
  • References
• 2: Conventional PCR
  • 2.1 Introduction
  • 2.2 Viruses and viroids
  • 2.3 Phytoplasmas
  • 2.4 Bacterial pathogens
  • 2.5 PCR detection of fungi
  • 2.6 Internal controls
  • 2.7 PCR optimization
    • 2.7.1 Primers
    • 2.7.2 Cycling conditions
    • 2.7.3 Reaction components
    • 2.7.4 PCR additives or enhancers
    • Protocols
      • Protocol 2.1: One-step PCR to detect potyviruses (Marie-Jeanne et al., 2000)
        • Materials
        • Method
        • Notes
      • Protocol 2.2: Nested-PCR to detect phytoplasmas
        • Materials
        • Method
        • Notes
  • References
• 3: Real-time PCR
  • 3.1 Introduction
  • 3.2 Coupling PCR amplification with the generation of fluorescence
  • 3.3 Probe-based real-time PCR
  • 3.4 Non-probe chemistries
  • 3.5 Reaction components and cycling programmes
  • 3.6 Real-time PCR instruments
  • 3.7 Selecting and designing an assay
    • 3.7.1 Choosing a target sequence
    • 3.7.2 Designing primers and probe
  • 3.8 Sample preparation and controls
  • 3.9 Assay performance and validation
  • 3.10 Qualitative and quantitative real-time PCR
  • 3.11 Multiplex real-time PCR
  • 3.12 Confirming the real-time PCR result
    • Protocols
      • Protocol 3.1: TaqMan real-time PCR detection of Erwinia amylovora using a portable SmartCycler® instrument (Cepheid)
        • Materials
        • Method
        • Notes
      • Protocol 3.2: One-step RT-qPCR (reverse transcription quantitative realtimePCR) for detection of Pepino mosaic virus (PepMV) in tomato seeds and in water using TaqMan-based chemistry
        • Materials
        • Method
        • Notes
      • Protocol 3.3: One-step RT-qPCR (reverse transcription quantitative real-time PCR) for detection and discrimination of Potato virus Y (PVY) isolates
        • Materials
        • Method
        • Notes
  • References
• 4: Loop-mediated isothermal amplification (LAMP)
  • 4.1 Introduction
  • 4.2 Loop-mediated isothermal amplification (LAMP)
  • 4.3 LAMP detection methods
  • 4.4 LAMP primer design
  • 4.5 Other isothermal methods
    • Protocols
      • Protocol 4.1: Loop-mediated isothermal amplification (LAMP) with end-point detection
        • Materials
        • Method
        • Notes
      • Protocol 4.2: Real-time LAMP
        • Materials
        • Method
  • References
• 5: DNA barcoding for identification of plant pathogens
  • 5.1 Introduction
  • 5.2 The application of DNA barcoding
  • 5.3 Technical considerations
    • 5.3.1 Sample collection and identification
    • 5.3.2 DNA extraction
    • 5.3.3 The sample and sample matrix
    • 5.3.4 Primer selection
    • 5.3.5 Polymerase selection
    • 5.3.6 Polymerase chain reaction (PCR)
    • 5.3.7 Whole genome amplification
    • 5.3.8 DNA sequencing
  • 5.4 Analytical considerations
    • 5.4.1 Analysis of sequence data
    • 5.4.2 Consensus sequences
    • 5.4.3 Analysing a DNA barcode
    • 5.4.4 Critical factors for evaluation of a DNA barcode
  • 5.5 Barcoding plant pathogens
    • 5.5.1 Bacteria
    • 5.5.2 Phytoplasmas
    • 5.5.3 Fungi
    • 5.5.4 European regulated plant pathogens
    • 5.5.5 Establishing a new barcoding protocol
    • Protocols
      • Protocol 5.1: Polymerase chain reaction and DNA sequencing
        • Materials
        • Method
      • Protocol 5.2: Sequence analysis and identification
        • Materials
        • Method
  • References
• 6: DNA barcoding of invertebrate plant pests
  • 6.1 Introduction
  • 6.2 Barcoding nematodes
    • 6.2.1 Genes and gene regions for molecular barcoding studies (biodiversity)
    • 6.2.2 Choice of barcode regions
    • 6.2.3 Barcoding as an identification tool
    • 6.2.4 Outlook for barcoding and identification
  • 6.3 Barcoding arthropods
  • 6.4 Conclusions and outlook
    • Protocols
      • Protocol 6.1: Generic DNA barcoding protocol for arthropods and nematodes
        • Method
        • Note
  • References
• 7: Microarrays
  • 7.1 Introduction
  • 7.2 Microarrays for pathogen detection
  • 7.3 Alternative microarray platforms
  • 7.4 Array design
  • 7.5 Conclusions
    • Protocols
      • Protocol 7.1: Glass slide array protocol for detection of RNA plant viruses
        • Materials
        • Method
        • Notes
      • Protocol 7.2: Array tube protocol
        • Materials
        • Method
        • Notes
  • References
• 8: Next-generation sequencing
  • 8.1 Introduction
  • 8.2 Total RNA sequence generation
    • 8.2.1 Sample extraction
    • 8.2.2 Library preparation
    • 8.2.3 Library normalization and pooling
    • 8.2.4 Sequencing
    • 8.2.5 Bioinformatics and analysis
  • 8.3 Quality control
    • 8.3.1 Why perform quality control?
    • 8.3.2 Typical QC protocol
  • 8.4 Metatranscriptomics for pathogen detection and viral whole genome sequencing
    • 8.4.1 Transcriptome assembly
    • 8.4.2 Sequence similarity searches
    • 8.4.3 Assigning taxonomy with MEGAN
    • Protocols
      • Protocol 8.1: Plant viral sequence generation using ScriptSeq libraries and an Illumina MiSeq
        • Materials
        • Methods
      • Protocol 8.2: Plant viral sequence analysis
        • Materials
        • Methods
  • References
• 9: On-site testing for plant pathogens
  • 9.1 Introduction
  • 9.2 On-site testing using antibody-based methods
  • 9.3 On-site nucleic acid extraction
  • 9.4 Real-time PCR in the field
  • 9.5 Isothermal amplification in the field
  • 9.6 Deployment of tests in the field
    • Protocols
      • Protocol 9.1: Real-time PCR using the Cepheid SmartCycler
        • Materials
        • Method
        • Note
      • Protocol 9.2: PEG extraction and real-time LAMP using the OptiGene Genie II
        • Materials
        • Method
        • Notes
  • References
• 10: Quality assurance for molecular testing in plant health
  • 10.1 Introduction
  • 10.2 Fit for purpose testing
  • 10.3 Selection of molecular tests
  • 10.4 Validation of molecular tests
    • 10.4.1 Validation and verification of molecular tests
    • 10.4.2 Performance characteristics and the validation process
    • 10.4.3 Revalidation
  • 10.5 Implementation of a molecular test in terms of quality
    • 10.5.1 Staff training
  • 10.6 Infrastructure and equipment
    • 10.6.1 Instrument calibration
  • 10.7 Critical reagents
  • 10.8 Measurement uncertainty
  • 10.9 Documentation
  • 10.10 Survey of test performance and quality management
    • 10.10.1 Quality controls
  • 10.11 Continuous improvement
  • 10.12 Conclusion
  • References
• Index