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 can—and cannot—be made when the ideal is not possible?

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Amplification of PR/RT Sequences

Reagent Preparation

SpecimenPreparation

Amplification Sequencing

Copy #

0 105 1010 1012

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Genotyping Assay Procedures

Patient virus

RNA Extraction

cDNA synthesis (RT)

cDNA amplification (PCR)

Electrophoresis

Data Analysis (base calling)

Report (sequence, mutations, interpretation)

Sequencing reactions

Positive and negative controls

Positive control

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LAB 3“1st Round PCR” Lab1st round amplification2nd round PCR set-up

Ideal Lab Workflow for Genotyping(Nested PCR/In-House Assays Only)

LAB 1"No Template" LabReagent storage and

Master mix preparation

LAB 2"Specimen Processing" Lab

Specimen receipt & storageRNA extraction and 1st round

PCR set-up

LAB 4"Post-amplification" Lab

2nd round amplificationGel electrophoresis

Sequencing reactionsCapillary electrophoresis

SPECIMENSFrozen plasmaWhole blood

DBS

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Example of a Four-Lab Layout (Nested PCR/In-House Assays Only)

-20°C freezer

wor

kben

ch

Ice machine

LAB 1

"No Template" Lab

LAB 2

"Specimen Processing" Lab

LAB 4

"Post-amplification" Lab

LAB 3

"Nested PCR" Lab/Amplification

Lab

-80°C

freezer

-20°C freezer

BSC

wor

kben

ch

BSC: bio-safety cabinet

Ice machine

Ice machine

-80°C

freezer-20°C

freezer

wor

kben

ch

Sequencer Gel electrophoresis area PCR machines

workbench PCR machines

-20°C freezer

Cen

trifu

ge

Pass-through window

Lab coat rack Lab coat rack

Lab coat rack Lab coat rack

NA extraction equipment

workbench

sink

sink

sink

sinkEye

wash

Eye wash

BSC

Cen

trifu

ge

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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

LAB 2"Amplification" Lab 1st round amplification

2nd round amplification

LAB 3"Post-amplification" Lab

Gel electrophoresisSequencing reactions

Capillary electrophoresis

Master mix preparation

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasmaWhole blood

DBS

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PCR Workstations (Dead-air Cabinets)

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Three-Lab SetupPre-Fabricated PCR Laboratory

http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1

123

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Three-Lab Setup (Option 2A)

LAB 1"No Template" LabReagent storage and

Master mix preparation

LAB 2"Specimen Processing and

PCR" LabSpecimen receipt & storageRNA extraction and 1st round

PCR set-up

LAB 3"Post-amplification" Lab

2nd round amplificationGel electrophoresis

Sequencing reactionsCapillary electrophoresis

1st round amplification2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasmaWhole blood

DBS

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Three-Lab Setup (Option 2B)

LAB 1"No Template" LabReagent storage and

Master mix preparation

LAB 2"Specimen Processing" Lab

Specimen receipt & storageRNA extraction and 1st round

PCR set-up

LAB 3"Post-amplification" Lab

2nd round amplificationGel electrophoresis

Sequencing reactionsCapillary electrophoresis

1st round amplification2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasmaWhole blood

DBS

Option 2B is the preferred configuration if no nested PCR

is performed

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Two-Lab Setup

LAB 1"Specimen Processing" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

LAB 2"Post-Amplification" Lab

1st round amplification

2nd round amplificationGel electrophoresis

Sequencing reactionsCapillary electrophoresis

Master mix preparation

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasmaWhole blood

DBS

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Example 2-Lab Layout

LAB 1

"Specimen Processing" Lab

LAB 2

"Post-amplification" Lab

-80°C

freezer

BSC: bio-safety cabinet

Ice machine

Ice machine

-80°C

freezer-20°C

freezer

wor

kben

ch

Sequencer Gel electrophoresis area PCR machines

Lab coat rack

Lab coat rack

NA extraction equipment

PCR machines

Dead air cabinet

Dea

d ai

r ca

bine

tC

entr

ifuge

sink Eye wash

-20°C freezer

BSC BSC

Eye wash

Cen

trifu

ge

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Discussion

What is the ideal set up for a molecular lab? What compromises can—and cannot—be

made when the ideal is not possible?

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other workflow considerationsWhat 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 doesn’t?

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Example 2: What works? What doesn’t?

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Example 3: What works? What doesn’t?

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Example 4: What works? What doesn’t?

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Example 5: What works? What doesn’t?

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Example 6: What works? What doesn’t?

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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