Polymerase Chain Reaction Quality Control and Quality Assurance Saran Grewal San Diego County Vector Control Program Vector 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.) EQUIPMENT Uninterrupted power supply, emergency backup generators, freezer temperature monitoring Maintenance Biosafety cabinets/still hoods Nucleic acid extraction and quantification systems Centrifuges Thermal cyclers Pipettes
THE ESSENTIALS REAGENTS Extraction and PCR kits UNG or UDG RNAse inhibitor Water Primers 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% Bleach 70% Ethanol Molecular grade water
VDDL WORKFLOW
THE METHODS SAMPLE COLLECTION AND PROCESSING NUCLEIC ACID EXTRACTION Positive and Negative Extraction Controls DNA/RNA QUALITY AND QUANTITY Organic contaminants and solvents Alcohol Phenol Guanidine isothiocyanate Urea Carbohydrates B mercaptoethanol Guanidinium from chaotropic lysis buffer STORAGE TEMPERATURE
THE METHODS (contd.) POLYMERASE CHAIN REACTION ASSAY (PCR) Primer design 18 28 nucleotides in length Avoid stretches of repeated nucleotides Aim 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 OPTIMIZATION Primer optimization Probe optimization Cycling parameters F-R Primer (nm) Probe (nm) 200 300 400 300-1000 300-1000/200 300-1000/300 300-1000/400 1000-1000 1000-1000/200 1000-1000/300 1000-1000/400 Ave Ct 20 19 18 17 16 15 16.80 17.09 Ave Ct 16 15.5 15 14.5 14 15.07 Forward Reverse Primer nm Final [ ] Forward Reverse/Probe nm Final [ ]
THE METHODS (contd.) PCR EFFICIENCY Ideally 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 EFFICIENCY GREATER THAN 110% POOR DNA OR RNA QUALITY REPURIFYING THE TEMPLATE HIGH TEMPLATE CONCENTRATION DILUTION CARRYOVER FROM NUCLEIC ACID PURIFICATION DILUTION/EXTRA DRYING TIME TO REMOVE ETHANOL LESS THAN 90% SUBOPTIMAL REAGENT CONCENTRATION ADJUSTING MAGNESIUM CONCENTRATION, PRIMER AND PROBE MATRIX SUBOPTIMAL THERMOCYCLING CONDITIONS ANNEALING TEMPERATURES ADJUSTED ACCORDING TO THE PRIMERS
THE METHODS (contd.) Monitoring the Positive Control
THE METHODS (contd.) SOFTWARE ANALYSIS SETTINGS AMPLIFICATION CURVE BASELINE LINEARITY AND C BASELINE RANGE SETTINGS
THE METHODS (contd.) SOFTWARE ANALYSIS SETTINGS THRESHOLD
THE METHODS (contd.) SOFTWARE ANALYSIS SETTINGS REFERENCE DYES Normalize 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 detection Provide a stable baseline for multiplex real time PCR and qrt PCR
INTERNAL QUALITY CONTROL PROCEDURES CONTROLS Extraction controls
INTERNAL QUALITY CONTROL PROCEDURES AMPLIFICATION CONTROLS Method Positive Control Inhibition controls (IPC)
INTERNAL QUALITY CONTROL PROCEDURES AMPLIFICATION CONTROLS Contamination controls Negative Extraction Control Method Negative Control Negative Template No Template Control
INTERNAL QUALITY CONTROL PROCEDURES AMPLIFICATION CONTROLS Contamination controls Positive Plasmid Control (Tracer)
INTERNAL QUALITY CONTROL PROCEDURES
INTERNAL QUALIY CONTROL PROCEDURES Ct CUTOFFS
DATA RECORDING, RECORD KEEPING, AND DATA EVALUATION Equipment Reagents, kits, primers and enzymes Sample processing and analyzing PCR data MSDS 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 Controls Not Setting the Baseline and Threshold Properly The Efficiency of the Reaction is Poor No documentation
Acknowledgements Dr. Nikos Gurfield Dr. Lynnie Cua Victoria Nguyen