Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications (eBook)

Foreword 5
Acknowledgements 7
Contents 8
1 Background 13
1.1 Aims of This Book 13
1.2 What Is PCR? 14
1.3 What Is the Use of PCR? 15
1.4 PCR and Infectious Diseases – The Veterinary Picture 16
1.5 Laboratory Diagnostic Technology 18
Bibliography 19
2 Traditional PCR 21
2.1 Traditional PCR 21
2.2 PCR Reaction 22
2.2.1 Primer Specifications 23
2.2.2 DNA Template 24
2.2.3 dNTPs 25
2.2.4 Magnesium Chloride 25
2.2.5 DNA Polymerase 25
2.2.6 Polymerase Buffer 26
2.2.7 Cycling Conditions 26
2.2.7.1 Initial Denaturation Step 26
2.2.7.2 Denaturation Step 26
2.2.7.3 Primer Annealing Step 27
2.2.7.4 Extension Step 27
2.2.7.5 PCR Amplification Cycle Number 27
2.2.7.6 Final Extension Step 28
2.3 PCR Set Up and Optimization 28
2.3.1 Optimizing a PCR Reaction 29
2.4 The PCR Plateau Effect 29
2.5 Radioisotope-PCR Based Methods 30
2.5.1 Radioisotopic-Based Methods 31
2.5.1.1 PCR Dot-Blot Assay 31
2.5.1.2 DNA Sequencing 32
Bibliography 34
3 Real-Time PCR The Basic Principles 38
3.1 Traditional PCR Versus Real Time PCR 38
3.1.1 PCR Kinetics 39
3.2 Optimising a Real-Time PCR Reaction 40
3.2.1 Primer Sets and Probe Design 40
3.2.1.1 Primer Secondary Structures 41
3.2.2 PCR Components and Assay Optimization 42
3.2.3 Real-Time Fluorescense Reporters 43
3.2.3.1 DNA Binding Dyes, SYBR® Green I 44
Advantages 44
Disadvantages 44
3.2.4 Melting Curve Dissociation Analysis 46
3.2.5 Probe-Based Chemistry 47
3.2.5.1 FRET-based Hydrolysis Probes, TaqMan Probes 47
3.2.5.2 TaqMan Probe Design Guidelines 48
3.2.5.3 MGB TaqMan Probe 48
3.2.6 FRET-Based Hybridisation Probes 49
3.2.6.1 Molecular Beacons 49
Advantages 49
Disadvantages 49
3.2.6.2 Probe Design 50
3.2.7 Scorpion Primers 51
Advantages 51
Disdvantages 51
3.2.7.1 Probe Design Specifications 51
3.2.8 LAMP 53
3.2.8.0 Advantages of LAMP LAMP 53
3.2.8.1 Primer Design 53
3.2.8.2 Primer Design Specifications 54
Bibliography 54
4 New Trends in the Diagnosis and Molecular Epidemiology of Viral Diseases 58
4.1 Background 59
4.1.1 Costs of Disease 59
4.1.2 Global Factors 60
4.1.3 Other Diseases 60
4.1.4 Major Problems 61
4.1.5 Need to Improve Diagnosis 61
4.1.6 Harmonization of Responses 62
4.1.7 Application of Various PCR Methods in Routine Diagnostic Virology 62
4.1.7.1 Gel-Based and Real-Time PCR Assays as Novel Tools 62
4.1.7.2 Various Real-Time PCR Assays Are Further Improving 63
4.1.7.3 Importance of Determining the Diagnostic Sensitivity and Specificity 64
4.1.7.4 The Simultaneous Use of Various Real-Time PCR Methods, Allowing 64
4.1.8 Multiplex PCR in Routine Diagnosis 65
4.1.9 Simultaneous Detection of Viruses and the Complex 65
4.1.10 Robots are Accelerating Molecular Diagnosis and Provide 66
4.1.11 Isothermal Amplification and the Use of Simple Thermo 66
4.1.12 Portable PCR Machines 67
4.1.13 Studies of Molecular Epidemiology 67
4.1.14 The OIE Rules for the International Standardization 68
4.1.15 OIE Manual of Diagnostic Tests and Vaccines 69
4.1.16 Validation and Quality Control of Polymerase Chain 69
4.2 PCR Methods Used in Routine Molecular Diagnostics 69
4.2.1 OIE Collaborating Center for the Biotechnology-Based 69
4.2.2 Recent Developments in the Field of Diagnostic Virology at 70
4.2.2.1 Improved Detection of Foot-and-Mouth Disease Virus (FMDV) 70
4.2.2.2 Solid Phase Microarrays in Veterinary Diagnostic Virology, Based 71
4.2.2.3 Padlock Probes for Broad-Range Detection and Subtyping of Avian 71
4.2.2.4 Novel TaqMan R and Primer-Probe Energy Transfer Assays 71
4.2.2.5 Development of a Real-Time PCR Assay Based on Primer-Probe 72
4.2.2.6 Simple and Rapid Detection of Swine Vesicular Disease Virus 72
4.2.2.7 Subtyping and Pathotyping of Avian Influenza Viruses 74
4.2.3 Ultra Rapid Nucleic Acid Amplification and Nucleotide Sequencing Analysis 74
4.2.4 Proximity Ligation, Novel Means of Protein Detection by Nucleic Acid Amplification 75
4.2.5 A Simple Magnetic Bead-Based Microarray for Detection and Discrimination of Pestiviruses 75
4.2.6 Detection of an Emerging Pestivirus in Cattle and Further 76
4.2.7 Molecular Epidemiology, New Approaches 77
4.2.8 Further Trends, New Directions in Molecular 77
4.2.8.1 Full-Genome Amplification of Viral Genomes 78
4.2.8.2 Full-Genome Sequencing of Viral Genomes 78
4.2.9 Viral Metagenomics, Search for Unknown Viruses 79
4.2.10 Summary and Recommendations 80
Bibliography 80
5 Disease Diagnosis Using Real-Time PCRSpecific Procedures for Important VeterinaryPathogens 83
SOP 1. Detection of Avian Influenza A Matrix Geneby Real Time TaqMan RT-PCR 86
1 Introduction 86
2 Materials 87
3 Procedure/Method 88
4 Results 89
5 Bibliography 91
6 Appendix 1 92
SOP 2. H7 Eurasian Real Time PCRs for the Detectionand Pathotyping of Eurasian H7 Avian Influenza Isolates 93
1 Introduction 93
2 Safety 94
3 Materials 94
4 Procedure/Method 97
5 Results 100
6 Bibliography 103
SOP 3. One Step RT PCR for Detection of H5 & H7 AvianInfluenza and Cleavage Site Sequencing
1 Introduction 104
2 Safety 104
3 Procedure/Method 105
4 Results 108
5 Bibliography 109
Appendix 1 110
SOP 4. Eurasian H5 Avian Influenza Real Time PCR 112
1 Introduction 112
2 Safety 112
3 Materials 113
4 Chemicals and Reagents 113
5 Procedure/Method 114
6 Results 116
7 Bibliography 118
SOP 5. Detection of Rift Valley Fever Virus by Real-TimeReverse Transcription-PCR 119
1 Detection of Rift Valley Fever Virus by Real-Time ReverseTranscription -PCR 119
2 RNA Isolation 119
3 RT-PCR Method 120
SOP 6. Swine Vesicular Disease (SVD) Virus One-StepRT-LAMP 123
1 Introduction 123
2 RNA Extraction 123
3 RT-LAMP 124
4 Appendix Primer Mixes 125
5 Abbreviations 125
SOP 7. Detection of African Swine Fever Virus DNA Using theIsothermal 127
1 Scope of SOP 127
2 Samples and DNA Extraction 128
3 Isothermal Reaction 128
4 Appendix. Oligos 129
SOP 8. Real-Time PCR Detection and Quantification of PorcineViruses Using Molecular Beacons 131
1 Introduction 131
2 Samples and DNA Extraction 131
3 Real-Time PCR 132
4 Appendix. Primers, Probes and Reaction Conditions 133
SOP 9. Swine Vesicular Disease Virus PriProET Two-StepReal-Time PCR 135
1 Introduction 135
2 RNA Extraction 136
3 cDNA Synthesis 136
4 Real-Time PCR 136
5 Appendix. Primer /Probe Mixes 137
SOP 10. Slope/End Point Analysis of Invader Data System 139
1 Introduction 139
2 Data Extraction 139
3 Determining Slope Rate 140
4 Statistical Analysis (End Point Analysis) 141
SOP 11. African Horse Sickness TaqMan RT-PCR 142
1 Introduction 142
2 RNA Extraction 142
3 Real-Time RT-PCR 143
4 Appendix. Primer /Probe Mixes 144
SOP 12. Bluetongue SYBR®-Green RT-PCR 146
1 Introduction 146
2 RNA Extraction 146
3 Real-Time RT-PCR 147
4 Appendix. Primer /Probe Mixes 148
SOP 13. BTV Serotype 4 SYBR® GREEN RT-PCR 150
1 Introduction 150
2 RNA Extraction 150
3 Real-Time RT-PCR 151
4 Appendix Primer /Probe Mixes 152
SOP 14. Real-Time Duplex Detection of Avian Influenza andNewcastle Disease Viruses 154
1 Introduction 154
2 RNA Extraction 154
3 Real-Time RT-PCR 155
SOP 15. Realtime RT PCR Detection of Influenza Virus MatrixGene Realtime RT PCR Detection of Velogenic NewcastleDisease Fusion Protein 157
1 Procedure 157
2 Procedure – Preparation of Stock Primer and Probes 162
3 Procedure- Realtime RT PCR Detection of Influenza VirusMatrix Gene, Realtime RT PCR Detection of VelogenicNewcastle Disease Fusion Protein Gene 163
4 Guidance Document- Assay Failure Response 177
SOP 16. Preparation of Silica Particles for Nucleic AcidExtraction 179
1 Introduction 179
2 Procedure 180
3 Bibliography 181
SOP 17. Boom-Silica RNA Extraction (GuSCN, phenol, Silica) 182
1 Introduction 182
2 Definition 182
3 Principle 182
4 Safety 183
5 Equipment 183
6 Reagents 184
7 Procedure 185
8 Bibliography 186
SOP 18. Mab Based Competitive ELISAs for H5 and H7Antibody Detection in Avian Sera 187
1 Introduction 187
2 Reagents 188
3 Biological Reagents 189
4 Assay Procedure 190
5 Evaluation of Results 192
SOP 19. Type A, H5, and H7 Avian Influenza Antigen DetectionELISAs 193
1 Introduction 193
2 Reagents 194
3 Biological Reagents 195
4 Assay Procedure 196
SOP 20. Ribonucleic Acid Extraction from Samples UsingTRIzol Reagent 198
1 Introduction 198
2 Risk assessment 198
3 Responsibilities 199
4 Materials 200
5 Chemicals 200
6 Reagents 200
7 Media 201
8 Organisms 201
9 Documentation 201
10 Procedure 201
11 Storage of Samples 202
12 Results 202
13 Troubleshooting 202
14 Bibliography 202
15. Tracking Sheet Used for Manual (TRIZOL) Extraction of RNA 203
SOP 21. Ambion Magnetic Beads Extraction (96-well) 204
1 Purpose 204
2 Scope 204
3 Definitions and Acronyms 204
4 Specimen Information 204
5 Reagents and Media 205
6 Supplies and Equipment 205
7 Special Safety Precautions 205
8 Equipment Calibration and Maintenance 205
9 Quality Control 205
10 Test Method Instructions 206
11 Calculations 208
12 Bibliography and Related Documentation 208
13 Appendices 208
14 Purpose 208
15 Procedure 209
SOP 22. Svanodip® FMDV-Ag Penside Test 210
1 Introduction 210
2 Contents 210
4 Interpretation of the Results 212
3 Procedure 211
SOP 24. Procedure for Using the Molecular Diagnostics Suite 216
1 Introduction 216
2 Purpose of SOP 216
3 Risk Assessment 216
4 Materials 218
5 Procedure 218
7 Maintenance 223
6 Results 223
8 Troubleshooting 223
9 Bibliography 224
SOP 23. FMDV PLA Assay 213
1 Introduction 213
2 Preparation of Proximity Probes 213
3 Assay Procedure 214
4 Interpretation of Results 215
SOP 25. One step TaqMan® RT-PCR for Diagnosis of FMDVand Related Vesicular Viruses 225
1 Introduction 225
2 Risk Assessment 225
3 Responsibilities 226
4 Materials 227
5 Procedure 229
6 Results 230
7 Bibliography 230
8 Tracking sheet used for manual (TRIZOL) extraction of RNA 231
SOP 26. Operation of the Stratagene Mx4000/Mx3005P forReal-Time PCR. One-Step RT-PCR Amplification of RNA fromVesicular Disease Viruses 232
1 Introduction 232
2 Risk Assessment 232
3 Responsibilities 233
4 Materials 233
5 Procedure (Alternative Procedures Are Described for theMx4000 and Mx3005P Machines) 234
6 Results 237
7 Analysis 238
8 Trouble Shooting 238
9 Bibliography 239
SOP 27. Differentiation of Sheep and Goat Poxviruses by RealTime PCR 240
1. Purpose 240
2. Scope 240
3. Samples and DNA Extraction 241
4. Real Time PCR 241
5. Results 243
6 Appendix 244
6 PCR Laboratory Set-up 245
6.1 Establishment of a PCR Laboratory 246
6.1.1 Minimum Layout Requirements for a Basic PCR Laboratory 246
6.1.2 Ideal Physical Arrangement for a PCR Laboratory 246
6.1.3 Ideal Physical Arrangement for a Real-Time PCR Laboratory 247
6.1.4 Reagent Preparation -- Area 1 247
6.1.4.1 Equipment Required in Area 1 247
6.1.4.2 Important Considerations Applicable to Area 1 248
6.1.5 DNA/RNA Extraction -- Area 2 248
6.1.5.1 Equipment Required in Area 2 249
6.1.5.2 Important Considerations Applicable to Area 2 249
6.1.6 Amplification (PCR) and Detection -- Area 3 250
6.1.6.1 Equipment Required in Area 3 250
6.1.6.2 Important Considerations Applicable to Area 3 250
6.1.7 Contamination and Sources 251
6.1.7.1 Amplicon Aerosols 251
6.1.7.2 Template Contaminants 251
6.1.7.3 Real Time PCR Systems and Contamination 252
6.1.8 Establishment of a PCR Assay 253
6.1.9 Validation of the Assay 253
6.1.9.1 Validation Pathway or Steps Followed for Assay Validation 253
6.2 Quality Assurance Programme or Accreditation 254
6.2.1 Proficiency Testing 255
6.2.2 PCR Controls 255
6.2.2.1 Positive Control Samples 255
6.2.2.2 Negative Control Samples 255
Bibliography 256
7 Analysis and Troubleshooting 257
7.1 How to Design Primers for Real-Time PCR Applications 257
7.1.1 TaqMan R Probes and Primer Design 259
7.1.2 Storage of Primers and TaqMan R Probes 260
7.1.3 SYBR R Green Assays 260
7.1.3.1 SYBR R Green I Behaviour During Real Time PCR 261
7.1.4 Optimisation of Primer Concentration 262
7.1.5 Multiple Bands on Gel or Multiple Peaks in the Melting Curve 263
7.1.6 Effect of Magnesium Chloride and Primer Concentration 264
7.1.7 Molecular Beacons Assays 264
7.2 Assay Performance Evaluation Using Standard Curves 264
7.2.1 Threshold selection 265
7.2.2 Quantification of Gene Targets with the Quantitative Real Time PCR: Absolute and Relative Gene Quantification 266
7.2.2.1 Absolute Quantification (Standard Curve Method) 266
7.2.3 Relative Quantification 266
7.2.3.1 Livak Method 2– CT Method 266
7.2.3.2 Pfaffl Method 267
7.2.3.3 Vandesompele Method 267
7.3 Most Common Problems When Performing Real-Time PCR 267
7.3.1 PCR Amplification Problems 267
7.3.2 Control Samples 268
7.3.2.1 Non Template Control (NTC) 268
7.3.2.2 Positive Controls 268
7.3.3 Signal Problems in Real Time PCR 268
7.3.4 Amplification Plots 269
7.4 Summary: Optimised Real-Time PCR Assay 270
Bibliography 271
8 Specifications for PCR Machines 274
Glossary of Terms 288
Index 316