16. Determine a Suitable Assay Protocol Quantitative PCR for CMV • Identify a suitable extraction method • Identify primers and probes from sequence database • Determine specificity of the primers and probes • Optimise reaction conditions • Determine analytical sensitivity of test (control) • Determine clinical sensitivity (clinical samples) • Laboratory evaluation (in parallel with existing method) • Document assay method and evaluation data
17. Determine a Suitable Assay Protocol Quality Control for the Assay NB: CONTAMINATION WITH PREVIOUSLY AMPLIFIED PCR PRODUCT IS THE MAJOR HAZARD Assay Controls -Positive - Negative (5 or 10%) - Environmental Internal QC - Swabs of work area - QC samples (sensitivity) External QC -QC samples (specificity and sensitivity
18.
19.
20.
21.
22.
23.
24. Introducing a Molecular Assay Involves 5 Steps Specimen Collection Nucleic Acid Extraction PCR Detection Reporting results An appropriate specimen must be collected from the correct site during the “clinical” phase of the disease process
27. CsCl gradient centrifugation Direction of migration • Centrifugation in CsCl which has high density • Sample is layered on top of CsCl gradient together with Etbr • Tube is centrifuged in ultra centrifuge (4hr at 300000g) • Particles separate through differences in their sedimentation rate (size & shape)
28. CsCl gradient centrifugation • Centrifugation in CsCl which has high density • Sample is layered on top of CsCl gradient together with Etbr • Tube is centrifuged in ultra centrifuge (4hr at 300000g) • Particles separate through differences in their sedimentation rate (size & shape) • NA of given sedimentation coefficient migrate down as a zone Direction of migration
29. Detection of product by agarose gel electrophoresis Bottom of tube is punctured 0.5 mL fractions collected