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

310 Pages·2010·6.46 MB·english

by  Erika Pestana

Early, Rapid and Sensitive Veterinary Molecular
Diagnostics - Real Time PCR Applications
· · ·
Ericka A. Pestana Sandor Belak Adama Diallo
·
John R. Crowther Gerrit J. Viljoen
Early, Rapid and Sensitive
Veterinary Molecular
Diagnostics - Real Time PCR
Applications
1 3
Dr.ErickaA.Pestana Prof.SandorBelak
DepartmentofNuclearSciences SwedishUniversityof
andApplications AgriculturalSciences
InternationalAtomicEnergy DepartmentofVirology
Agency(IAEA) SE-75007Uppsala
A1400Vienna,Austria Sweden
[email protected] [email protected]
Dr.AdamaDiallo Dr.JohnR.Crowther
DepartmentofNuclearSciences DepartmentofNuclearSciences
andApplications andApplications
InternationalAtomicEnergy InternationalAtomicEnergy
Agency(IAEA) Agency(IAEA)
A1400Vienna,Austria A1400Vienna,Austria
[email protected] [email protected]
Prof.GerritJ.Viljoen
DepartmentofNuclearSciences
andApplications
InternationalAtomicEnergy
Agency(IAEA)
A1400Vienna,Austria
[email protected]
ISBN978-90-481-3131-0 e-ISBN978-90-481-3132-7
DOI10.1007/978-90-481-3132-7
SpringerDordrechtHeidelbergLondonNewYork
LibraryofCongressControlNumber:2009938825
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Foreword
The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is
involved in agricultural research and development and assists Member States of
FAO and IAEA in improving strategies to ensure food security through the use
of nuclear techniques and related biotechnologies, where such techniques have a
valuableandoftenuniquerole.Inparticular,nuclearandnuclearrelatedmolecular
diagnosticmethodshaverapidlyevolvedinthepast20years,sincetheadventofthe
Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural
areas such as, improving soil and water management; producing better crop vari-
eties;diagnosingplantandanimaldiseases;controllinginsectpestsandimproving
foodqualityandsafety.
The uses of nucleic acid-directed methods have increased significantly in the
past 5 years and have made important contributions to disease control country
programmes for improving national and international trade. These developments
include the more routine use of PCR, Real-Time PCR and PCR-Sequencing as
diagnosticandcharacterizationtoolsinveterinarydiagnosticlaboratories.However,
therearemanyproblems associatedwiththetransferandparticularly,theapplica-
tion of this technology. These include lack of consideration of the establishment
ofquality-assuredprocedures,therequiredset-upofthelaboratoryandtheproper
trainingofstaff.Thiscanleadtoasituationwhereresultsarenotassured.
This book gives a comprehensive account of the practical aspects of real-time
PCRandstrongconsiderationisgiventoensureitsoptimaluseinadiagnosticlabo-
ratory environment. This includes the basic principles, setting-up of a Real-Time
PCR laboratory; Good Laboratory Practice and Standard Operating Procedures;
Diagnostic Implementation, Execution and Interpretation, Analysis and Problem
Solving.ExamplesofStandardOperatingProceduresasusedinindividualspecial-
ist laboratories and an outline of training materials necessary for Real-Time PCR
technology transfer are presented. The difficulties, advantages and disadvantages
inPCRandReal-TimePCRapplicationsareexplainedandplacedincontextwith
othertestsystems.
Emphasis is placed on the use of Real-Time PCR for detection of pathogens,
withaparticularfocusondiagnosticiansandscientistsfromthedevelopingworld.
v
vi Foreword
Itishopedthatthisbookwillenablereadersfromvariousdisciplinesandlevelsof
expertise to better judge the merits of early and rapid nuclear and nuclear related
moleculardiagnosticapproachesandtoincreasetheirskillsandknowledgeinorder
toassistinamorelogical,efficientandassureduseofthesetechnologies.
LiangQu
Director:
JointFAO/IAEADivisionofNuclearTechniquesinFoodandAgriculture
DepartmentofNuclearSciencesandApplications
Acknowledgements
The authors would like to thank the following people and Institutions for their
contributions,suggestionsandpermissions:
Donald King and the Institute of Animal Health, Pirbright Laboratory, United
Kingdom; Sharon Hietala and the California Animal Health and Food Safety
Laboratory; Ian Brown and the Veterinary Laboratories Agency, Surrey-United
Kingdom; Janusz Paweska and the Special Pathogens Unit (NICD-NHLS) in
Sandringham-Johannesburg, for their valuable contributions on specific proce-
duresfordiseasediagnostics.Bio-Rad,RocheDiagnosticsGmbH;PEBiosystems;
AppliedBiosystems;PerkinElmer,andStratagene;fortheir“visual”help.
Thanks to the people at the SVA-SLU Virology R&D Division, at the MOD
section of SVA VIP and at Uppsala University (the groups of Professors Jonas
BlombergandUlfLandegren).
Many thanks to Anne-Lie Blomstram et al. and Lihong Liu et al. (see refs) for
providingfigures.
TheauthorsalsowouldliketoacknowledgeSVAandSLU,FORMAS,AGRIA,
KBM, as well as the European FP6 and FP7 projects and their research consortia:
LAB-ON-SITE(SSP3-513Â645),CSF&WILD-BOAR(SSP1-501599),EPIZONE
(FOOD-CT-2006-016236),FLUTESTandFLUTRAIN.
Special thanks to Marcus Neusser from Bio-Rad, for his advice and help on
completingthisventure,andtoallthosewhopeerreviewedthisbook.
vii
Contents
1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 AimsofThisBook . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 WhatIsPCR? . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 WhatIstheUseofPCR? . . . . . . . . . . . . . . . . . . . . . 3
1.4 PCRandInfectiousDiseases–TheVeterinaryPicture . . . . . . 4
1.5 LaboratoryDiagnosticTechnology . . . . . . . . . . . . . . . . 6
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 TraditionalPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1 TraditionalPCR . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 PCRReaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.1 PrimerSpecifications . . . . . . . . . . . . . . . . . . . 11
2.2.2 DNATemplate . . . . . . . . . . . . . . . . . . . . . . 12
2.2.3 dNTPs . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2.4 MagnesiumChloride . . . . . . . . . . . . . . . . . . . 13
2.2.5 DNAPolymerase . . . . . . . . . . . . . . . . . . . . . 13
2.2.6 PolymeraseBuffer . . . . . . . . . . . . . . . . . . . . 14
2.2.7 CyclingConditions . . . . . . . . . . . . . . . . . . . . 14
2.3 PCRSetUpandOptimization . . . . . . . . . . . . . . . . . . 16
2.3.1 OptimizingaPCRReaction . . . . . . . . . . . . . . . 17
2.4 ThePCRPlateauEffect . . . . . . . . . . . . . . . . . . . . . . 17
2.5 Radioisotope-PCRBasedMethods . . . . . . . . . . . . . . . . 18
2.5.1 Radioisotopic-BasedMethods . . . . . . . . . . . . . . 19
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3 Real-TimePCR–TheBasicPrinciples . . . . . . . . . . . . . . . . 27
3.1 TraditionalPCRVersusRealTimePCR . . . . . . . . . . . . . 27
3.1.1 PCRKinetics . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 OptimisingaReal-TimePCRReaction . . . . . . . . . . . . . . 29
3.2.1 PrimerSetsandProbeDesign . . . . . . . . . . . . . . 29
3.2.2 PCRComponentsandAssayOptimization. . . . . . . . 31
3.2.3 Real-TimeFluorescenseReporters . . . . . . . . . . . . 32
3.2.4 MeltingCurveDissociationAnalysis. . . . . . . . . . . 35
3.2.5 Probe-BasedChemistry . . . . . . . . . . . . . . . . . . 36
ix
x Contents
3.2.6 FRET-BasedHybridisationProbes . . . . . . . . . . . . 38
3.2.7 ScorpionPrimers . . . . . . . . . . . . . . . . . . . . . 40
3.2.8 LAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4 NewTrendsintheDiagnosisandMolecularEpidemiology
ofViralDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.1.1 CostsofDisease . . . . . . . . . . . . . . . . . . . . . 48
4.1.2 GlobalFactors . . . . . . . . . . . . . . . . . . . . . . 49
4.1.3 OtherDiseases . . . . . . . . . . . . . . . . . . . . . . 49
4.1.4 MajorProblems . . . . . . . . . . . . . . . . . . . . . . 50
4.1.5 NeedtoImproveDiagnosis . . . . . . . . . . . . . . . . 50
4.1.6 HarmonizationofResponses . . . . . . . . . . . . . . . 51
4.1.7 ApplicationofVariousPCRMethodsinRoutine
Diagnostic Virology . . . . . . . . . . . . . . . . . . . 51
4.1.8 MultiplexPCRinRoutineDiagnosis . . . . . . . . . . . 54
4.1.9 Simultaneous Detection of Viruses and the
ComplexDiagnosis,Developmentof“Multi
PCR”AssaysSimplifyDiagnosis . . . . . . . . . . . . 54
4.1.10 RobotsareAcceleratingMolecularDiagnosis
andProvideBetterSafety . . . . . . . . . . . . . . . . . 55
4.1.11 IsothermalAmplificationandtheUseofSimple
ThermoBlocksCanReplaceCostlyPCRMachines . . . 55
4.1.12 PortablePCRMachines. . . . . . . . . . . . . . . . . . 56
4.1.13 StudiesofMolecularEpidemiology . . . . . . . . . . . 56
4.1.14 The OIE Rules for the International
StandardizationandValidationofthePCR-Based
DiagnosticAssays . . . . . . . . . . . . . . . . . . . . 57
4.1.15 OIEManualofDiagnosticTestsandVaccines
forTerrestrialAnimals2004,2008 . . . . . . . . . . . . 58
4.1.16 ValidationandQualityControlofPolymerase
ChainReactionMethodsUsedfortheDiagnosis
ofInfectiousDiseases(ChapterI.1.4.oftheOIEManual) 58
4.2 PCRMethodsUsedinRoutineMolecularDiagnostics. . . . . . 58
4.2.1 OIECollaboratingCenterfortheBiotechnology-
Based Diagnosis of Infectious Diseases in
VeterinaryMedicine . . . . . . . . . . . . . . . . . . . 58
4.2.2 RecentDevelopmentsintheFieldofDiagnostic
VirologyattheOIECollaboratingCenterforthe
BiotechnologybasedDiagnosisofInfectious
DiseasesinVeterinaryMedicine . . . . . . . . . . . . . 59
4.2.3 UltraRapidNucleicAcidAmplificationand
NucleotideSequencingAnalysis . . . . . . . . . . . . . 63
Contents xi
4.2.4 ProximityLigation,NovelMeansofProtein
DetectionbyNucleicAcidAmplification . . . . . . . . 64
4.2.5 ASimpleMagneticBead-BasedMicroarrayfor
DetectionandDiscriminationofPestiviruses . . . . . . 64
4.2.6 DetectionofanEmergingPestivirusinCattle
and Further Characterization by Means of
MolecularDiagnosticsandReverseGenetics . . . . . . 65
4.2.7 MolecularEpidemiology,NewApproaches . . . . . . . 66
4.2.8 FurtherTrends,NewDirectionsinMolecular
DiagnosticVirology. . . . . . . . . . . . . . . . . . . . 66
4.2.9 ViralMetagenomics,SearchforUnknownViruses . . . 68
4.2.10 SummaryandRecommendations . . . . . . . . . . . . . 69
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5 Disease Diagnosis Using Real-Time PCR Specific
ProceduresforImportantVeterinaryPathogens . . . . . . . . . . . 73
SOP1. DetectionofAvianInfluenzaAMatrixGeneby
RealTimeTaqManRT-PCR . . . . . . . . . . . . . . . . . 76
SOP2. H7EurasianRealTimePCRsfortheDetectionand
PathotypingofEurasianH7AvianInfluenzaIsolates . . . . 83
SOP3. OneStepRTPCRforDetectionofH5&H7Avian
InfluenzaandCleavageSiteSequencing . . . . . . . . . . . 94
SOP4. EurasianH5AvianInfluenzaRealTimePCR . . . . . . . . 102
SOP5. DetectionofRiftValleyFeverVirusbyReal-Time
ReverseTranscription-PCR . . . . . . . . . . . . . . . . . 109
SOP6. SwineVesicularDisease(SVD)VirusOne-StepRT-LAMP 113
SOP7. DetectionofAfricanSwineFeverVirusDNAUsing
theIsothermal . . . . . . . . . . . . . . . . . . . . . . . . 117
SOP8. Real-TimePCRDetectionandQuantificationof
PorcineVirusesUsingMolecularBeacons. . . . . . . . . . 121
SOP9. SwineVesicularDiseaseVirusPriProETTwo-Step
Real-TimePCR . . . . . . . . . . . . . . . . . . . . . . . . 125
SOP10. Slope/EndPointAnalysisofInvaderDataSystem . . . . . 129
SOP11. AfricanHorseSicknessTaqManRT-PCR . . . . . . . . . . 132
(cid:2)
SOP12. BluetongueSYBRR-GreenRT-PCR . . . . . . . . . . . . 136
(cid:2)
SOP13. BTVSerotype4SYBRR GREENRT-PCR . . . . . . . . . 140
SOP14. Real-TimeDuplexDetectionofAvianInfluenzaand
NewcastleDiseaseViruses . . . . . . . . . . . . . . . . . . 144
SOP15. RealtimeRTPCRDetectionofInfluenzaVirus
Matrix Gene Realtime RT PCR Detection of
VelogenicNewcastleDiseaseFusionProtein . . . . . . . . 147
SOP16. PreparationofSilicaParticlesforNucleicAcidExtraction . 169
SOP17. Boom-SilicaRNAExtraction(GuSCN,phenol,Silica) . . . 172
SOP18. MabBasedCompetitiveELISAsforH5andH7
AntibodyDetectioninAvianSera . . . . . . . . . . . . . . 177