HIV Drug Resistance Training

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HIV Drug Resistance Training

• Module 4
• Molecular Lab Set-up and Workflow

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Topics

• Principles of Lab Set Up
• Other Workflow Considerations
• Application of Principles

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Objectives

• Identify principles for laboratory setup and
  workflow design that prevents lab contamination.
• Identify examples of good/poor practices in lab
  setup and workflow.
• Given the current lab setup, identify changes
  that must be made to prevent lab contamination.

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principles of lab set up

• What is the ideal set up for a molecular lab?
• What compromises canand cannotbe made when the
  ideal is not possible?

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Amplification of PR/RT Sequences
Reagent Preparation
SpecimenPreparation
Amplification
Sequencing
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Genotyping Assay Procedures
Patient virus
RNA Extraction
cDNA synthesis (RT)
cDNA amplification (PCR)
Sequencing reactions
Electrophoresis
Data Analysis (base calling)
Report (sequence, mutations, interpretation)
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Ideal Lab Workflow for Genotyping(Nested
PCR/In-House Assays Only)
LAB 2 "Specimen Processing" Lab Specimen receipt
storage RNA extraction and 1st round PCR set-up
SPECIMENS Frozen plasma Whole blood DBS
LAB 4 "Post-amplification" Lab 2nd round
amplification Gel electrophoresis Sequencing
reactions Capillary electrophoresis
LAB 1 "No Template" Lab Reagent storage
and Master mix preparation
LAB 3 1st Round PCR Lab 1st round
amplification 2nd round PCR set-up
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Example of a Four-Lab Layout (Nested
PCR/In-House Assays Only)
Ice machine
sink
Centrifuge
LAB 2 "Specimen Processing" Lab
LAB 1 "No Template" Lab
workbench
Pass-through window
workbench
NA extraction equipment
sink
Ice machine
Eye wash
Lab coat rack
Lab coat rack
Lab coat rack
Lab coat rack
Eye wash
Ice machine
workbench
sink
workbench
LAB 4 "Post-amplification" Lab
LAB 3 "Nested PCR" Lab/Amplification Lab
Centrifuge
Sequencer
Gel electrophoresis area
sink
PCR machines
PCR machines
workbench
BSC bio-safety cabinet
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Three-Lab Setup (Option 1)
LAB 1 "Specimen Processing" Lab Specimen receipt
storage RNA extraction and 1st round PCR
set-up
SPECIMENS Frozen plasma Whole blood DBS
Master mix preparation
LAB 3 "Post-amplification" Lab Gel
electrophoresis Sequencing reactions Capillary
electrophoresis
LAB 2 "Amplification" Lab 1st round
amplification 2nd round amplification
2nd round PCR set-up
In dead-air cabinet with dedicated pipettes
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PCR Workstations (Dead-air Cabinets)
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Three-Lab SetupPre-Fabricated PCR Laboratory
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Three-Lab Setup (Option 2A)
LAB 2 "Specimen Processing and PCR" Lab Specimen
receipt storage RNA extraction and 1st round
PCR set-up
SPECIMENS Frozen plasma Whole blood DBS
1st round amplification 2nd round PCR set-up
LAB 1 "No Template" Lab Reagent storage
and Master mix preparation
LAB 3 "Post-amplification" Lab 2nd round
amplification Gel electrophoresis Sequencing
reactions Capillary electrophoresis
In dead-air cabinet with dedicated pipettes
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Three-Lab Setup (Option 2B)
SPECIMENS Frozen plasma Whole blood DBS
LAB 2 "Specimen Processing" Lab Specimen receipt
storage RNA extraction and 1st round PCR set-up
LAB 1 "No Template" Lab Reagent storage
and Master mix preparation
LAB 3 "Post-amplification" Lab 2nd round
amplification Gel electrophoresis Sequencing
reactions Capillary electrophoresis
1st round amplification 2nd round PCR set-up
Option 2B is the preferred configuration if no
nested PCR is performed
In dead-air cabinet with dedicated pipettes
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Two-Lab Setup
LAB 1 "Specimen Processing" Lab Specimen receipt
storage RNA extraction and 1st round PCR
set-up
SPECIMENS Frozen plasma Whole blood DBS
Master mix preparation
LAB 2 "Post-Amplification" Lab 1st round
amplification 2nd round amplification Gel
electrophoresis Sequencing reactions Capillary
electrophoresis
2nd round PCR set-up
In dead-air cabinet with dedicated pipettes
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Example 2-Lab Layout
Ice machine
Centrifuge
LAB 1 "Specimen Processing" Lab
Dead air cabinet
NA extraction equipment
sink
Eye wash
Lab coat rack
Lab coat rack
Eye wash
Ice machine
PCR machines
Dead air cabinet
workbench
LAB 2 "Post-amplification" Lab
Centrifuge
Sequencer
Gel electrophoresis area
PCR machines
BSC bio-safety cabinet
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Discussion

• What is the ideal set up for a molecular lab?
• What compromises canand cannotbe made when the
  ideal is not possible?

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other workflow considerations

• What else can we do to minimize the chances of
  contamination in the lab?

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Personnel

• Perform pre-amplification work before entering
  post-amplification areas
• Incorporate this (and all related
  contamination-control policies) into SOPs and/or
  lab policies

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Lab Coats

• Disposable or dedicated for each area
• Color-coded if possible

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SOP

• Make copies of SOPs available in each lab to
  prevent upstream traffic

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Storage Freezer Locations

• Specimen logs, access to computerized inventory,
  etc. in pre-amplification area
• RT-PCR reagents should be stored in separate
  freezers from specimens.

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application of principles

• What are examples of good and poor workflow?
• What can we do, in our labs, to get the best
  possible lab set up and workflow?

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Example 1 What works? What doesnt?
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Example 2 What works? What doesnt?
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Example 3 What works? What doesnt?
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Example 4 What works? What doesnt?
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Example 5 What works? What doesnt?
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Example 6 What works? What doesnt?
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Discussion

• What are examples of good and poor workflow?
• What can we do, in our labs, to get the best
  possible lab set up and workflow?

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Small Group Activity

• What can we do, in our labs, to get the best
  possible lab set up and workflow?

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Summary

• Principles of Lab Set Up
• Other Workflow Considerations
• Application of Principles