Polymerase Chain Reaction Quality Control and Quality ...POLYMERASE CHAIN REACTION ASSAY (PCR)...
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Polymerase Chain Reaction Quality Control and Quality Assurance Saran Grewal San Diego County Vector Control Program Vector Disease and Diagnostic Laboratory
Transcript of Polymerase Chain Reaction Quality Control and Quality ...POLYMERASE CHAIN REACTION ASSAY (PCR)...
Polymerase Chain ReactionQuality Control and Quality Assurance
Saran Grewal San Diego County Vector Control ProgramVector Disease and Diagnostic Laboratory
How Positive is Positive?
TOPICS
THE LABORATORY(GENERAL LABORATORY PRACTICES FOR QUALITY ASSURANCE)
THE ESSENTIALS(REAGENTS AND CONSUMABLES)
THE METHODS(ANALYTICAL PROCEDURES AND ASSESSMENT)
INTERNAL QUALITY CONTROL PROCEDURES(UNG,POSITVE AND NEGATIVE CONTROLS FOR EXTRACTION AND AMPLIFICATION,PLASMID CONSTRUCTS, IPC, AMPLIFICATION EFFICIENCY)
THE PROOF(DATA RECORDING, RECORD KEEPING, AND DATA EVALUATION)
THE LABORATORY
TRAINING OF PERSONNEL
THE LABORATORY
STANDARD OPERATING PROCEDURES (SOP)PERSONAL PROTECTIVE EQUIPMENT(PPE)
THE LABORATORY (contd.)
FACILITY DESIGN
Master mix room
Nucleic acid extraction room
Amplification room
Signs
WORKFLOW
Unidirectional
THE LABORATORY (contd.)
EQUIPMENTUninterrupted power supply, emergency backup generators, freezer temperature monitoring
MaintenanceBiosafety cabinets/Still hoodsNucleic acid extraction and quantification systemsCentrifugesThermal cyclersPipettes
THE ESSENTIALS
REAGENTSExtraction and PCR kitsUNG or UDGRNAse inhibitorWaterPrimers and probes
Storage
DISPOSABLES Pipette tips: barrier tips, aerosol‐resistant PCR tubes and plates: certified RNase and DNase free Sample tubes
THE ESSENTIALS (contd)
Laboratory cleaning
10% Bleach70% Ethanol
Molecular grade water
VDDL WORKFLOW
THE METHODSSAMPLE COLLECTION AND PROCESSINGNUCLEIC ACID EXTRACTIONPositive and Negative Extraction Controls
DNA/RNA QUALITY AND QUANTITYOrganic contaminants and solvents
AlcoholPhenolGuanidine isothiocyanateUreaCarbohydratesB‐mercaptoethanolGuanidinium from chaotropic lysis buffer
STORAGE TEMPERATURE
THE METHODS (contd.)
POLYMERASE CHAIN REACTION ASSAY (PCR)
Primer design18–28 nucleotides in lengthAvoid stretches of repeated nucleotidesAim for 50% GC content, which helps to prevent mismatch stabilization
Choose that primers have compatible Tms (within 5°C of each other and 10°C less than the probe
Avoid sequence complementarity between all primers employed in an assay and within each primer
Pick the probe first and no G at the 5’ end
THE METHODS (contd.)
PCR OPTIMIZATIONPrimer optimizationProbe optimizationCycling parameters
16.80 17.09
151617181920
Ave Ct
Forward‐ Reverse Primer nM Final [ ]
F-R Primer (nM)
Probe (nM)200 300 400
300-1000 300-1000/200 300-1000/300 300-1000/400
1000-10001000-1000/200 1000-1000/300 1000-1000/400
15.07
14
14.5
15
15.5
16Av
e Ct
Forward‐ Reverse/Probe nM Final [ ]
THE METHODS (contd.)
PCR EFFICIENCYIdeally the efficiency (E) of a PCR should be 100%, meaning that for each cycle the amount of product doubles (E=2) For an efficiency of 100%, the slope is ‐3.32. A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope between ‐3.58 and ‐3.10.
Amplification Efficiency = [10(-1/-slope) – 1] x 100
THE METHODS (contd.)
CAUSES OF POOR EFFICIENCYGREATER THAN 110%POOR DNA OR RNA QUALITY HIGH TEMPLATE CONCENTRATIONCARRYOVER FROM NUCLEIC ACID PURIFICATION
LESS THAN 90%SUBOPTIMAL REAGENT CONCENTRATION
SUBOPTIMAL THERMOCYCLING CONDITIONS
REPURIFYING THE TEMPLATE
ADJUSTING MAGNESIUM CONCENTRATION, PRIMER AND PROBE MATRIX
DILUTION/EXTRA DRYING TIME TO REMOVE ETHANOL
ANNEALING TEMPERATURES ADJUSTED ACCORDING TO THE PRIMERS
DILUTION
THE METHODS (contd.)
Monitoring the Positive Control
THE METHODS (contd.)
SOFTWARE ANALYSIS SETTINGSAMPLIFICATION CURVE BASELINE LINEARITY AND C BASELINE RANGE SETTINGS
THE METHODS (contd.)
SOFTWARE ANALYSIS SETTINGSTHRESHOLD
THE METHODS (contd.)
SOFTWARE ANALYSIS SETTINGSREFERENCE DYESNormalize for non‐PCR related fluctuations in fluorescence (e.g., caused by pipetting errors)
Normalize for fluctuations in fluorescence resulting from machine “noise”Compensate for variations in instrument excitation and detectionProvide a stable baseline for multiplex real‐time PCR and qRT‐PCR
INTERNAL QUALITY CONTROL PROCEDURES
CONTROLS
Extraction controls
INTERNAL QUALITY CONTROL PROCEDURES
AMPLIFICATION CONTROLSMethod Positive ControlInhibition controls (IPC)
INTERNAL QUALITY CONTROL PROCEDURES
AMPLIFICATION CONTROLSContamination controlsNegative Extraction ControlMethod Negative Control
Negative Template No Template Control
INTERNAL QUALITY CONTROL PROCEDURES
AMPLIFICATION CONTROLSContamination controlsPositive Plasmid Control (Tracer)
INTERNAL QUALITY CONTROL PROCEDURES
INTERNAL QUALIY CONTROL PROCEDURES
Ct CUTOFFS
DATA RECORDING, RECORD KEEPING, AND DATA EVALUATION
EquipmentReagents, kits, primers and enzymesSample processing and analyzingPCR dataMSDS Labelling
How positive is positive?
LESSONS LEARNED
Poor Primer and Probe Design Inappropriate Quality and Quantity of DNA and RNA
Not Using Appropriate Reagents Introducing Cross‐Contamination Not Using a Appropriate ControlsNot Setting the Baseline and Threshold Properly The Efficiency of the Reaction is Poor No documentation
Acknowledgements
Dr. Nikos Gurfield Dr. Lynnie CuaVictoria Nguyen
