HIV Drug Resistance Training

1. HIV Drug Resistance Training Module 4: Molecular Lab Set-up and Workflow

2. Topics • Principles of Lab Set Up • Other Workflow Considerations • Application of Principles

3. 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.

4. principles of lab set up What is the ideal set up for a molecular lab? What compromises can—and cannot—be made when the ideal is not possible?

5. Amplification of PR/RT Sequences Reagent Preparation SpecimenPreparation Sequencing Amplification 0 1012 1010 105 Copy #

6. Positive and negative controls Positive control Genotyping Assay Procedures Patient virus RNA Extraction cDNA synthesis (RT) cDNA amplification (PCR) Sequencing reactions Electrophoresis Data Analysis (base calling) Report (sequence, mutations, interpretation)

7. Ideal Lab Workflow for Genotyping(Nested PCR/In-House Assays Only) SPECIMENS Frozen plasma Whole blood DBS LAB 2 "Specimen Processing" Lab Specimen receipt & storage RNA extraction and 1st round PCR set-up 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

8. -20°C freezer -20°C freezer -20°C freezer -80°C freezer -80°C freezer -20°C freezer BSC BSC 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 sink workbench 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

9. 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

10. PCR Workstations (Dead-air Cabinets)

11. Three-Lab SetupPre-Fabricated PCR Laboratory 3 2 1 http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1

12. 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

13. 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

14. 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

15. -80°C freezer -20°C freezer -20°C freezer -80°C freezer BSC BSC 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

16. Discussion • What is the ideal set up for a molecular lab? • What compromises can—and cannot—be made when the ideal is not possible?

17. other workflow considerations What else can we do to minimize the chances of contamination in the lab?

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

19. Lab Coats • Disposable or dedicated for each area • Color-coded if possible

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

21. 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.

22. 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?

23. Example 1: What works? What doesn’t?

24. Example 2: What works? What doesn’t?

25. Example 3: What works? What doesn’t?

26. Example 4: What works? What doesn’t?

27. Example 5: What works? What doesn’t?

28. Example 6: What works? What doesn’t?

29. 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?

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

31. Summary • Principles of Lab Set Up • Other Workflow Considerations • Application of Principles