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

1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Aims ofThisBook . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 What IsPCR? . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 What Is theUseofPCR? . . . . . . . . . . . . . . . . . . . . . 3 1.4 PCRand InfectiousDiseases–TheVeterinaryPicture . . . . . . 4 1.5 Laboratory Diagnostic Technology . . . . . . . . . . . . . . . . 6 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Traditional PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.1 Traditional PCR . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.2 PCRReaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2.1 Primer Specifications . . . . . . . . . . . . . . . . . . . 11 2.2.2 DNATemplate . . . . . . . . . . . . . . . . . . . . . . 12 2.2.3 dNTPs . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2.4 Magnesium Chloride . . . . . . . . . . . . . . . . . . . 13 2.2.5 DNAPolymerase . . . . . . . . . . . . . . . . . . . . . 13 2.2.6 PolymeraseBuffer . . . . . . . . . . . . . . . . . . . . 14 2.2.7 CyclingConditions . . . . . . . . . . . . . . . . . . . . 14 2.3 PCRSetUp andOptimization . . . . . . . . . . . . . . . . . . 16 2.3.1 Optimizing aPCRReaction . . . . . . . . . . . . . . . 17 2.4 ThePCRPlateauEffect . . . . . . . . . . . . . . . . . . . . . . 17 2.5 Radioisotope-PCR Based Methods . . . . . . . . . . . . . . . . 18 2.5.1 Radioisotopic-Based Methods . . . . . . . . . . . . . . 19 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3 Real-Time PCR – The Basic Principles . . . . . . . . . . . . . . . . 27 3.1 Traditional PCR Versus Real Time PCR . . . . . . . . . . . . . 27 3.1.1 PCRKinetics . . . . . . . . . . . . . . . . . . . . . . . 28 3.2 OptimisingaReal-TimePCRReaction . . . . . . . . . . . . . . 29 3.2.1 PrimerSets andProbeDesign . . . . . . . . . . . . . . 29 3.2.2 PCR Components and Assay Optimization . . . . . . . . 31 3.2.3 Real-Time Fluorescense Reporters . . . . . . . . . . . . 32 3.2.4 Melting Curve Dissociation Analysis . . . . . . . . . . . 35 3.2.5 Probe-Based Chemistry . . . . . . . . . . . . . . . . . . 36 ix x Contents 3.2.6 FRET-Based Hybridisation Probes . . . . . . . . . . . . 38 3.2.7 Scorpion Primers . . . . . . . . . . . . . . . . . . . . . 40 3.2.8 LAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 4 New Trends in the Diagnosis and Molecular Epidemiology of Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.1.1 Costs ofDisease . . . . . . . . . . . . . . . . . . . . . 48 4.1.2 Global Factors . . . . . . . . . . . . . . . . . . . . . . 49 4.1.3 OtherDiseases . . . . . . . . . . . . . . . . . . . . . . 49 4.1.4 MajorProblems . . . . . . . . . . . . . . . . . . . . . . 50 4.1.5 Need to Improve Diagnosis . . . . . . . . . . . . . . . . 50 4.1.6 Harmonization of Responses . . . . . . . . . . . . . . . 51 4.1.7 Application of Various PCR Methods in Routine Diagnostic Virology . . . . . . . . . . . . . . . . . . . 51 4.1.8 Multiplex PCR in Routine Diagnosis . . . . . . . . . . . 54 4.1.9 Simultaneous Detection of Viruses and the Complex Diagnosis, Development of “Multi PCR” Assays Simplify Diagnosis . . . . . . . . . . . . 54 4.1.10 Robots are Accelerating Molecular Diagnosis andProvideBetterSafety . . . . . . . . . . . . . . . . . 55 4.1.11 Isothermal Amplification and the Use of Simple Thermo Blocks Can Replace Costly PCR Machines . . . 55 4.1.12 Portable PCR Machines . . . . . . . . . . . . . . . . . . 56 4.1.13 Studies of Molecular Epidemiology . . . . . . . . . . . 56 4.1.14 The OIE Rules for the International Standardization and Validation of the PCR-Based Diagnostic Assays . . . . . . . . . . . . . . . . . . . . 57 4.1.15 OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2004, 2008 . . . . . . . . . . . . 58 4.1.16 Validation and Quality Control of Polymerase Chain Reaction Methods Used for the Diagnosis of Infectious Diseases (Chapter I.1.4. of the OIE Manual) 58 4.2 PCR Methods Used in Routine Molecular Diagnostics . . . . . . 58 4.2.1 OIE Collaborating Center for the Biotechnology- Based Diagnosis of Infectious Diseases in VeterinaryMedicine . . . . . . . . . . . . . . . . . . . 58 4.2.2 Recent Developments in the Field of Diagnostic Virology at the OIE Collaborating Center for the Biotechnology based Diagnosis of Infectious Diseases inVeterinaryMedicine . . . . . . . . . . . . . 59 4.2.3 Ultra Rapid Nucleic Acid Amplification and Nucleotide Sequencing Analysis . . . . . . . . . . . . . 63 Contents xi 4.2.4 Proximity Ligation, Novel Means of Protein Detection byNucleicAcidAmplification . . . . . . . . 64 4.2.5 A Simple Magnetic Bead-Based Microarray for Detection andDiscriminationofPestiviruses . . . . . . 64 4.2.6 Detection of an Emerging Pestivirus in Cattle and Further Characterization by Means of Molecular Diagnostics and Reverse Genetics . . . . . . 65 4.2.7 Molecular Epidemiology, New Approaches . . . . . . . 66 4.2.8 Further Trends, New Directions in Molecular Diagnostic Virology . . . . . . . . . . . . . . . . . . . . 66 4.2.9 Viral Metagenomics, Search for Unknown Viruses . . . 68 4.2.10 Summary and Recommendations . . . . . . . . . . . . . 69 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 5 Disease Diagnosis Using Real-Time PCR Specific Procedures for Important Veterinary Pathogens . . . . . . . . . . . 73 SOP 1. Detection of Avian Influenza A Matrix Gene by RealTimeTaqManRT-PCR . . . . . . . . . . . . . . . . . 76 SOP 2. H7 Eurasian Real Time PCRs for the Detection and Pathotyping of Eurasian H7 Avian Influenza Isolates . . . . 83 SOP 3. One Step RT PCR for Detection of H5 & H7 Avian Influenza and Cleavage Site Sequencing . . . . . . . . . . . 94 SOP 4. Eurasian H5 Avian Influenza Real Time PCR . . . . . . . . 102 SOP 5. Detection of Rift Valley Fever Virus by Real-Time ReverseTranscription-PCR . . . . . . . . . . . . . . . . . 109 SOP 6. Swine Vesicular Disease (SVD) Virus One-Step RT-LAMP 113 SOP 7. Detection of African Swine Fever Virus DNA Using the Isothermal . . . . . . . . . . . . . . . . . . . . . . . . 117 SOP 8. Real-Time PCR Detection and Quantification of Porcine Viruses Using Molecular Beacons . . . . . . . . . . 121 SOP 9. Swine Vesicular Disease Virus PriProET Two-Step Real-TimePCR . . . . . . . . . . . . . . . . . . . . . . . . 125 SOP 10. Slope/End Point Analysis of Invader Data System . . . . . 129 SOP 11. African Horse Sickness TaqMan RT-PCR . . . . . . . . . . 132 SOP 12. Bluetongue SYBR R  -GreenRT-PCR . . . . . . . . . . . . 136 SOP 13. BTV Serotype 4 SYBR R  GREEN RT-PCR . . . . . . . . . 140 SOP 14. Real-Time Duplex Detection of Avian Influenza and NewcastleDiseaseViruses . . . . . . . . . . . . . . . . . . 144 SOP 15. Realtime RT PCR Detection of Influenza Virus Matrix Gene Realtime RT PCR Detection of Velogenic Newcastle Disease Fusion Protein . . . . . . . . 147 SOP 16. Preparation of Silica Particles for Nucleic Acid Extraction . 169 SOP 17. Boom-Silica RNA Extraction (GuSCN, phenol, Silica) . . . 172 SOP 18. Mab Based Competitive ELISAs for H5 and H7 Antibody Detection in Avian Sera . . . . . . . . . . . . . . 177 xii Contents SOP 19. Type A, H5, and H7 Avian Influenza Antigen DetectionELISAs . . . . . . . . . . . . . . . . . . . . . . 183 SOP 20. Ribonucleic Acid Extraction from Samples Using TRIzol Reagent . . . . . . . . . . . . . . . . . . . . . . . . 188 SOP 21. Ambion Magnetic Beads Extraction (96-well) . . . . . . . . 194 SOP 22. Svanodip R  FMDV-Ag Penside Test . . . . . . . . . . . . . 200 SOP23. FMDVPLAAssay . . . . . . . . . . . . . . . . . . . . . . 203 SOP 24. Procedure for Using the Molecular Diagnostics Suite . . . . 206 SOP 25. One step TaqMan R  RT-PCR for Diagnosis of FMDVandRelatedVesicularViruses . . . . . . . . . . . . 215 SOP 26. Operation of the Stratagene Mx4000/Mx3005P for Real-Time PCR. One-Step RT-PCR Amplification ofRNAfromVesicularDiseaseViruses . . . . . . . . . . . 222 SOP 27. Differentiation of Sheep and Goat Poxviruses by RealTimePCR . . . . . . . . . . . . . . . . . . . . . . . . 230 6 PCR Laboratory Set-up . . . . . . . . . . . . . . . . . . . . . . . . 235 6.1 Establishment of a PCR Laboratory . . . . . . . . . . . . . . . 236 6.1.1 Minimum Layout Requirements for a Basic PCR Laboratory . . . . . . . . . . . . . . . . . . . . . . . . 236 6.1.2 Ideal Physical Arrangement for a PCR Laboratory . . . . 236 6.1.3 Ideal Physical Arrangement for a Real-Time PCR Laboratory . . . . . . . . . . . . . . . . . . . . . . 237 6.1.4 Reagent Preparation – Area 1 . . . . . . . . . . . . . . . 237 6.1.5 DNA/RNAExtraction –Area 2 . . . . . . . . . . . . . . 238 6.1.6 Amplification (PCR) andDetection –Area 3 . . . . . . . 240 6.1.7 Contamination and Sources . . . . . . . . . . . . . . . . 241 6.1.8 Establishment of aPCRAssay . . . . . . . . . . . . . . 243 6.1.9 Validation of theAssay . . . . . . . . . . . . . . . . . . 243 6.2 Quality Assurance Programme or Accreditation . . . . . . . . . 244 6.2.1 Proficiency Testing . . . . . . . . . . . . . . . . . . . . 245 6.2.2 PCRControls . . . . . . . . . . . . . . . . . . . . . . . 245 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 7 Analysis and Troubleshooting . . . . . . . . . . . . . . . . . . . . . 247 7.1 HowtoDesignPrimers forReal-TimePCRApplications . . . . 247 7.1.1 TaqMan R Probes and Primer Design . . . . . . . . . . . 249 7.1.2 Storage of Primers and TaqMan R Probes . . . . . . . . 250 7.1.3 SYBR R GreenAssays . . . . . . . . . . . . . . . . . . 250 7.1.4 Optimisation of Primer Concentration . . . . . . . . . . 252 7.1.5 Multiple Bands on Gel or Multiple Peaks in the Melting Curve . . . . . . . . . . . . . . . . . . . 253 7.1.6 Effect of Magnesium Chloride and Primer Concentration 254 7.1.7 Molecular Beacons Assays . . . . . . . . . . . . . . . . 254 7.2 Assay Performance Evaluation Using Standard Curves . . . . . 254 7.2.1 ThresholdSelection . . . . . . . . . . . . . . . . . . . . 255 Contents xiii 7.2.2 Quantification of Gene Targets with the Quantitative Real Time PCR: Absolute and RelativeGeneQuantification . . . . . . . . . . . . . . . 256 7.2.3 RelativeQuantification . . . . . . . . . . . . . . . . . . 256 7.3 Most Common Problems When Performing Real-Time PCR . . 257 7.3.1 PCRAmplificationProblems . . . . . . . . . . . . . . . 257 7.3.2 ControlSamples . . . . . . . . . . . . . . . . . . . . . 258 7.3.3 Signal Problems in Real Time PCR . . . . . . . . . . . 258 7.3.4 AmplificationPlots . . . . . . . . . . . . . . . . . . . . 259 7.4 Summary:OptimisedReal-TimePCRAssay . . . . . . . . . . . 260 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 8 Specifications for PCR Machines . . . . . . . . . . . . . . . . . . . 265 Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307