Lecture 2 diagnostic molecular microbiology bls

Diagnosis of Infectious Disease

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[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

detecting the microbe detecting the response

Direct Detection ..going to the source

Applied Molecular Microbiology

MOLECULAR DIAGNOSTIC TESTING IN THE MICROBIOLOGY LABORATORY ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Evaluate the Need ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Evaluate the Need ,[object Object],[object Object],[object Object],[object Object],[object Object]

Evaluate the Need ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Identify the changes to be introduced ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Determine a Suitable Assay Protocol ,[object Object],[object Object],[object Object],[object Object]

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

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

Provide Adequate Resources ,[object Object],[object Object],Work Flow in MDU Reagent Preparation Specimen Extraction Amplification Detection Specimen Addition ,[object Object],[object Object],1 2 Low DNA Level 3 High DNA Level 4 Low DNA Level 5 Clean Room

Provide Adequate Resources ,[object Object],[object Object],[object Object]

[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Provide Adequate Resources

Educate the Clients ,[object Object],[object Object],There will be changes in - result interpretation - test costs - specimen requirements - turn-around times

Evaluate Long-term Performance ,[object Object],[object Object],[object Object],[object Object],[object Object]

Continuous Improvements ,[object Object],[object Object],[object Object],[object Object],[object Object]

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

Purification and Isolation of Nucleic Acids

Purification and Isolation of Nucleic Acids ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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)

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

Detection of product by agarose gel electrophoresis Bottom of tube is punctured 0.5 mL fractions collected

Purification and Isolation of Nucleic Acids ,[object Object],[object Object],[object Object],Disadvantages • Time consuming/labour intensive • Involves the use of noxious chemicals • Phenol oxidises DNA/RNA resulting in loss of target NA Other Methods • Anion exchange chromatography • Boom process (silica particles)

Purification and Isolation of Nucleic Acids Advantages of the Boom method • Fast • No dangerous chemicals used • High recovery of pure NA • Can be used for both DNA and RNA ,[object Object],[object Object],[object Object]

Detection and Characterisation of DNA ,[object Object],[object Object],Direct Methods • Agarose gel electrophoresis • Pulse field gel electrophoresis • Restriction fragment length polymorphism • Capillary electrophoresis • Hybridisation with NA probes Indirect Methods • After amplification of NA target • Real-time detection

DIRECT METHODS OF DETECTING NUCLEIC ACIDS

Pulsed Field Gel Electrophoresis ,[object Object],[object Object]

PFGE Profile of Acinetobacter Isolates ,[object Object],[object Object]

Hybridisation Analysis ,[object Object],[object Object],[object Object]

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

Hybridisation Analysis ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Southern blotting – large DNA fragments ,[object Object],[object Object],[object Object],[object Object]

Lecture 2 diagnostic molecular microbiology bls

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Lecture 2 diagnostic molecular microbiology bls