HIV Drug Resistance Training Module 4 Molecular Lab

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

Topics § § § Principles of Lab Set Up Other Workflow Considerations Application of Principles 2

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. § 3

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? 4

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

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

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 1 st round PCR set-up LAB 4 "Post-amplification" Lab LAB 1 "No Template" Lab Reagent storage and Master mix preparation LAB 3 “ 1 st Round PCR” Lab 2 nd round amplification Gel electrophoresis Sequencing reactions Capillary electrophoresis 1 st round amplification 2 nd round PCR set-up 7

-20°C freezer BSC workbench Ice machine Lab coat rack "Specimen Processing" Lab NA extraction equipment Centrifuge PCR machines Lab coat rack Eye wash sink Eye wash workbench Ice machine LAB 4 -20°C freezer "Nested PCR" Lab/Amplification Lab workbench Lab coat rack sink -80°C freezer "No Template" Lab workbench BSC LAB 2 Passthrough window LAB 3 sink Ice machine LAB 1 Lab coat rack -20°C freezer -80°C freezer workbench sink Centrifuge Example of a Four-Lab Layout (Nested PCR/In-House Assays Only) "Postamplification" Lab Sequencer Gel electrophoresis area PCR machines 8 BSC: bio-safety cabinet

Three-Lab Setup (Option 1) SPECIMENS Frozen plasma Whole blood DBS LAB 1 "Specimen Processing" Lab Specimen receipt & storage Master mix preparation RNA extraction and 1 st round PCR set-up LAB 3 "Post-amplification" Lab LAB 2 "Amplification" Lab Gel electrophoresis Sequencing reactions Capillary electrophoresis 1 st round amplification 2 nd round PCR set-up 2 nd round amplification In dead-air cabinet with dedicated pipettes 9

PCR Workstations (Dead-air Cabinets) 10

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

Three-Lab Setup (Option 2 A) SPECIMENS Frozen plasma Whole blood DBS LAB 2 "Specimen Processing and PCR" Lab Specimen receipt & storage RNA extraction and 1 st round PCR set-up 1 st round amplification 2 nd round PCR set-up LAB 1 "No Template" Lab Reagent storage and Master mix preparation LAB 3 "Post-amplification" Lab 2 nd round amplification Gel electrophoresis Sequencing reactions Capillary electrophoresis In dead-air cabinet with dedicated pipettes 12

Three-Lab Setup (Option 2 B) SPECIMENS Frozen plasma Whole blood DBS LAB 2 "Specimen Processing" Lab Specimen receipt & storage RNA extraction and 1 st round PCR set-up LAB 1 "No Template" Lab Reagent storage and Master mix preparation Option 2 B is the preferred configuration if no nested PCR is performed LAB 3 "Post-amplification" Lab 1 st round amplification 2 nd round PCR set-up 2 nd round amplification Gel electrophoresis Sequencing reactions Capillary electrophoresis In dead-air cabinet with dedicated pipettes 13

Two-Lab Setup SPECIMENS Frozen plasma Whole blood DBS LAB 1 "Specimen Processing" Lab Specimen receipt & storage Master mix preparation RNA extraction and 1 st round PCR set-up LAB 2 "Post-Amplification" Lab 1 st round amplification 2 nd round PCR set-up 2 nd round amplification Gel electrophoresis Sequencing reactions Capillary electrophoresis 14 In dead-air cabinet with dedicated pipettes

Ice machine -20°C freezer BSC -80°C freezer Centrifuge Example 2 -Lab Layout "Specimen Processing" Lab NA extraction equipment Lab coat rack PCR machines Eye wash Dead air cabinet Ice machine LAB 2 -20°C freezer Centrifuge workbench Eye wash sink -80°C freezer Dead air cabinet LAB 1 "Postamplification" Lab Sequencer Gel electrophoresis area BSC: bio-safety cabinet PCR machines 15

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

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

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

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

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

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. § 21

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? 22

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

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

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

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

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

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

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? 29

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

Summary ü ü ü Principles of Lab Set Up Other Workflow Considerations Application of Principles 31