Traditional methods of diagnosing infectious diseases like isolation, serology, and direct detection have limitations that influence their clinical utility. Molecular methods using nucleic acid detection and characterization have advantages like faster results, increased sensitivity and specificity, and ability to adapt to new instrumentation. Introducing molecular diagnostic testing requires evaluating needs, identifying suitable changes, developing protocols, providing resources, educating staff, and ensuring continuous quality improvement.

1. Diagnosis of Infectious Disease

4. detecting the microbe detecting the response

5. Direct Detection ..going to the source

6. Applied Molecular Microbiology

9. MOLECULAR DIAGNOSTICS

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

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

25. Purification and Isolation of Nucleic Acids

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

34. DIRECT METHODS OF DETECTING NUCLEIC ACIDS

39. DNA Hybridisation 5’-AAAGGGTTACGAACGACGCC-3’ 3’-TTTCCCAATGCTTGCTGCGG-5’ Double Stranded DNA Target DNA