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Background	p. 1
Aims of This Book	p. 1
What Is PCR?	p. 2
What Is the Use of PCR?	p. 3
PCR and Infectious Diseases - The Veterinary Picture	p. 4
Laboratory Diagnostic Technology	p. 6
Bibliography	p. 7
Traditional PCR	p. 9
Traditional PCR	p. 9
PCR Reaction	p. 10
Primer Specifications	p. 11
DNA Template	p. 12
dNTPs	p. 13
Magnesium Chloride	p. 13
DNA Polymerase	p. 13
Polymerase Buffer	p. 14
Cycling Conditions	p. 14
PCR Set Up and Optimization	p. 16
Optimizing a PCR Reaction	p. 17
The PCR Plateau Effect	p. 17
Radioisotope-PCR Based Methods	p. 18
Radioisotopic-Based Methods	p. 19
Bibliography	p. 22
Real-Time PCR-The Basic Principles	p. 27
Traditional PCR Versus Real Time PCR.	p. 27
PCR Kinetics	p. 28
Optimising a Real-Time PCR Reaction	p. 29
Primer Sets and Probe Design	p. 29
PCR Components and Assay Optimization	p. 31
Real-Time Fluorescense Reporters	p. 32
Melting Curve Dissociation Analysis	p. 35
Probe-Based Chemistry	p. 36
FRET-Based Hybridisation Probes	p. 38
Scorpion Primers	p. 40
LAMP	p. 42
Bibliography	p. 43
New Trends in the Diagnosis and Molecular Epidemiology at Viral Diseases	p. 47
Background	p. 48
Costs of Disease	p. 48
Global Factors	p. 49
Other Diseases	p. 49
Major Problems	p. 50
Need to Improve Diagnosis	p. 50
Harmonization of Responses	p. 51
Application of Various PCR Methods in Routine Diagnostic Virology	p. 51
Multiplex PCR in Routine Diagnosis	p. 54
Simultaneous Detection of Viruses and the Complex Diagnosis, Development of “Multi PCR” Assays Simplify Diagnosis	p. 54
Robots are Accelerating Molecular Diagnosis and Provide Better Safety	p. 55
Isothermal Amplification and the Use of Simple Thermo Blocks Can Replace Costly PCR Machines	p. 55
Portable PCR Machines	p. 56
Studies of Molecular Epidemiology	p. 56
The OIE Rules for the International Standardization and Validation of the PCR-Based Diagnostic Assays	p. 57
OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2004, 2008	p. 58
Validation and Quality Control of Polymerase Chain Reaction Methods Used for the Diagnosis of Infectious Diseases (Chapter I1.4. of the OIE Manual)	p. 58
PCR Methods Used in Routine Molecular Diagnostics	p. 58
OIE Collaborating Center for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine	p. 58
Recent Developments in the Field of Diagnostic Virology at the OIE Collaborating Center for the Biotechnology based Diagnosis of Infections Diseases in Veterinary Medicine	p. 59
Ultra Rapid Nucleic Acid Amplification and Nucleotide Sequencing Analysis	p. 63
Proximity Ligation, Novel-Means of Protein Detection by Nucleic Acid Amplification	p. 64
A Simple Magnetic Bead-Based Microarray for Detection and Discrimination of Pestiviruses	p. 64
Detection of an Emerging Pestivirus in Cattle and Further Characterization by Means of Molecular Diagnostics and Reverse Genetics	p. 65
Molecular Epidemiology, New Approaches	p. 66
Further Trends, New Directions in Molecular Diagnostic Virology	p. 66
Viral Metagenomics, Search for Unknown Viruses	p. 68
Summary and Recommendations	p. 69
Bibliography	p. 69
Disease Diagnosis Using Real-Time PCR Specific Procedures for Important Veterinary Pathogens	p. 73
Detection of Avian Influenza A Matrix Gene by Real Time TaqMan RT-PCR	p. 76
H7 Eurasian Real Time PCRs for the Detection and Pathotyping of Eurasian H7 Avian Influenza Isolates	p. 83
One Step RT PCR for Detection of H5 & H7 Avian Influenza and Cleavage Site Sequencing	p. 94
Eurasian H5 Avian Influenza Real Time PCR	p. 102
Detection of Rift Valley Fever Virus by Real-Time Reverse Transcription-PCR	p. 109
Swine Vesicular Disease (SVD) Virus One-Step RT-LAMP	p. 113
Detection of African Swine Fever Virus DNA Using the Isothermal	p. 117
Real-Time PCR Detection and Quantification of Porcine Viruses Using Molecular Beacons	p. 121
Swine Vesicular Disease Virus PriProET Two-Step Real-Time PCR	p. 125
Slope/End Point Analysis of Invader Data System	p. 129
African Horse Sickness TaqMan RT-PCR	p. 132
Bluetongue SYBR®-Green RT-PCR	p. 136
BTV Serotype 4 SYBR® GREEN RT-PCR	p. 140
Real-Time Duplex Detection of Avian Influenza and Newcastle Disease Viruses	p. 144
Realtime RT PCR Detection of Influenza Virus Matrix Gene Realtime RT PCR Detection of Velogenic Newcastle Disease Fusion Protein	p. 147
Preparation of Silica Particles for Nucleic Acid Extraction	p. 169
Boom-Silica RNA Extraction (GuSCN, phenol, Silica)	p. 172
Mab Based Competitive ELISAs for H5 and H7 Antibody Detection in Avian Sera	p. 177
Type A, H5, and H7 Avian Influenza Antigen Detection ELlSAs	p. 183
Ribonucleic Acid Extraction from Samples Using TRIzol Reagent	p. 188
Ambion Magnetic Beads Extraction (96-well)	p. 194
Svanodip® FMDV-Ag Penside Test	p. 200
FMDV PLA Assay	p. 203
Procedure for Using the Molecular Diagnostics Suite	p. 206
One step TaqMan® RT-PCR for Diagnosis of FMDV and Related Vesicular Viruses	p. 215
Operation of the Stratagene Mx4000/Mx3005P for Real-Time PCR. One-Step RT-PCR Amplification of RNA from Vesicular Disease Viruses	p. 222
Differentiation of Sheep and Goat Poxviruses by Real Time PCR	p. 230
PCR Laboratory Set-up	p. 235
Establishment of a PCR Laboratory	p. 236
Minimum Layout Requirements for a Basic PCR Laboratory	p. 236
Ideal Physical Arrangement for a PCR Laboratory	p. 236
Ideal Physical Arrangement for a Real-Time PCR Laboratory	p. 237
Reagent Preparation - Area 1	p. 237
DNA/RNA Extraction - Area 2	p. 238
Amplification (PCR) and Detection - Area 3	p. 240
Contamination and Sources	p. 241
Establishment of a PCR Assay	p. 243
Validation of the Assay	p. 243
Quality Assurance Programme or Accreditation	p. 244
Proficiency Testing	p. 245
PCR Controls	p. 245
Bibliography	p. 246
Analysis and Troubleshooting	p. 247
How to Design Primers for Real-Time PCR Applications	p. 247
TaqMan® Probes and Primer Design	p. 249
Storage of Primers and TaqMan® Probes	p. 250
SYBR® Green Assays	p. 250
Optimisation of Primer Concentration	p. 252
Multiple Bands on Gel or Multiple Peaks in the Melting Curve	p. 253
Effect of Magnesium Chloride and Primer Concentration	p. 254
Molecular Beacons Assays	p. 254
Assay Performance Evaluation Using Standard Curves	p. 254
Threshold Selection	p. 255
Quantification of-Gene-Targets with the Quantitative Real Time PCR: Absolute and Relative Gene Quantification	p. 256
Relative Quantification	p. 256
Most Common Problems When Performing Real-Time PCR	p. 257
PCR Amplification Problems	p. 257
Control Samples	p. 258
Signal Problems m Real Time PCR	p. 258
Amplification Plots	p. 259
Summary: Optimised Real-Time PCR Assay	p. 260
Bibliography	p. 261
pecifications for PCR Machines	p. 265
Glossary of Terms	p. 279
Index	p. 307
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Early, Rapid and Sensitive Veterinary Molecular Diagnostics - Real Time Pcr Applications

9048131316

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