Molecular Diagnostics – How To Get Started

Danny L. Wiedbrauk, Ph.D.

Warde Medical Laboratory

Ann Arbor, Michigan

Molecular Diagnostics

Fastest growing area in laboratory medicine.

Increasing numbers of: Detection technologies Commercial detection kits Analyte specific reagents Instrumentation options

Growth ProjectionsTest Category $ Millions (2001) Est. Growth/Year

Coagulation 690 0 - 3%

Clinical Chemistry 3,485 1 – 3%

Microbiology 1,540 1 – 2%

Hematology 1,215 1 – 3%

Immunoassay 4,155 2 – 5%

Whole Blood Glucose 3,770 10 – 15%

Flow Cytometry 590 10 – 15%

Point of Care (POCT) 725 10 – 12%

Molecular Diagnostics 805 25 – 35%

Cook, SC. Advance for Administrators of the Laboratory, 2007;16(8):56

Driving Forces for Change Nucleic acid detection methods have

become an integral and necessary component of laboratory medicine.

Newer technologies are making molecular diagnostic procedures available to a wider range of clinical laboratories than ever before.

Traditional PCR Methods

ReagentPrep

SamplePrep

End-pointPCR

Analysis

Detection

Traditional PCR Methods

Complex master mixes and amplification profiles

Involved handling amplified nucleic acids Had increased potential for producing

false positive results Procedures took 1-3 days Many procedures had subjective

interpretations

Real-Time PCR –The Enabling Technology

What’s So Cool About Real-Time PCR?

Decreased turnaround times Simultaneous amplification, detection, and data analysis

Closed system No additions made after specimen is added Contamination control

Numerical vs. visual readouts Less subjectivity Able to monitor amplification efficiency

What’s So Cool About Real-Time PCR?

More automation Some to include automated sample preparation

FDA approved methods Training and technical support Decreased QA/QC costs

What is Real-Time PCR?

Cycle Number

PCR Amplification PlotF

luor

esce

nce

(R

n)

Threshold CT

Plateau

Baseline

Exponential Phase

Linear Phase

Dilution Series

1-108 virus copies

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61

Cycle number

Re

lati

ve

flu

ore

sce

nc

e

+ Control

Serum

Urine

CSF

Cycle Number

TaqMan RT-PCR ResultsFor Enterovirus

What This Means to You

These new technologies allow general laboratories such as Microbiology and Hematology to do nucleic acid testing.

How do we get started?

Contamination Control

Contamination Control

Target and Probe Amplification tests create millions of new replication-competent molecules.

These amplified nucleic acids (amplicons) can contaminate gloves, skin, clothing, and the environment.

Contamination of a specimen with one amplicon is sufficient to produce a false-positive reaction.

Contamination Control

DNA is very hardy and is resistant to many traditional decontamination procedures including: Alcohols and organic solvents Detergents and disinfectants Autoclaving Drying

What Works?

10% bleach (freshly made) Ultraviolet light Nucleolytic agents Some acids

Contamination Control

Engineering controls Procedural controls Chemical controls within the assay Surveillance

Engineering Controls

Reagent Preparation

Specimen Preparation

Reaction Setup andAmplification

Product Analysis

Dedicated Hallway

Work flow

Engineering Controls - Kits

Reagent Preparation

Specimen PreparationAmplification Setup

Amplification,Detection, and Product Analysis

Work flow

Real-Time PCR

Reagent Preparation

Specimen PreparationAmplification Setup inBiosafety Cabinet

Amplification,Detection, and Product Analysis

Work flow

Disposables/Procedural Controls

Aerosol resistant tips Separate lab coats/gloves Dedicated equipment Unidirectional workflow Only one tube open Low level positive controls

Procedural Controls

Dedicated equipment Separate lab

coats/gloves

Surveillance

Wipe testing Monitoring negative controls Monitoring prevalence rates

Question

How can you do wipe testing in a lab that routinely grows the infectious agent?

Wipe Testing

Wipe testing can reinforce the need to disinfect the environment daily.

Can improve infection control in lab

What Skills Do You Need?

Ability to multiple things at once Fastidious work habits Able to accurately pipette small fluid volumes High tolerance for change Excellent communication skills Outstanding customer service ethic

What Will You Be Doing?

Extracting nucleic acids Some type of amplification technology Signal or nucleic acid detection Interpretation of results

Extraction Systems

Remove inhibitory and interfering substances without significantly altering the amount or quality of the target nucleic acid

Available Chemistries

Liquid extraction and salting out Gentra Systems PureGene Orca Research IsoQuick

Nonspecific binding of nucleic acids to a solid phase DEAE Dextran Silica

Spin Column TechnologySpin Column Technology

LysisLysis BindBind WashWash EluteElute

AA BB CC DD

Qiagen Spin ColumnsQiagen Spin Columns

Silica membrane plus Silica membrane plus chaotropic saltschaotropic salts

No organic extractionNo organic extraction No ethanol precipitation No ethanol precipitation DNA or RNA, BothDNA or RNA, Both Wide range of clinical samples Wide range of clinical samples Mini, midi, and maxi columnsMini, midi, and maxi columns

QIAcube Spin-Column Processing

Commercial Spin Columns

Qiagen Clontech Amresco Gentra Systems Stratagene Roche Molecular

QIAmp, Rneasy Nucleospin Cyclo-Prep Generation Capture Strata Prep High Pure

Magnetic Particle Technology

Magnetic Particle Kits

Cortex Biochem Promega Dynal

Roche Molecular

MegaZorb

MagnaSil

Dynabeads

DNA Direct

DNA Isolation Kit

Magnetic Separators

BioMerieux Mini MagExtractorBioMerieux Mini MagExtractor

Semi-automated extractor for fewer specimens

Magnetic silica particles Recovery of RNA and DNA

from different sample types 50 l eluate 24 samples in 1h

Qiagen BioRobot EZ1Qiagen BioRobot EZ1

Fully automatedFully automated Pre-programmed protocol Pre-programmed protocol

cardscards Prefilled, sealed reagent Prefilled, sealed reagent

cartridgescartridges Small footprintSmall footprint 1-6 samples in 15-30 min1-6 samples in 15-30 min 200 to 350 200 to 350 l of sample l of sample Variety of samplesVariety of samples

Qiagen BioRobot EZ1Qiagen BioRobot EZ1

MagNA Pure Compact SystemMagNA Pure Compact System

Fully automated Barcoded prefilled, sealed

reagent cartridges Variable sample types (100

to 1000 l) 50 to 200 l elution volumes 1 to 8 isolations per run Benchtop; small footprint

Major Clinical Systems for Real-Time PCR

ABI 7300/7500

Roche LightCycler

Cepheid SmartCycler Cepheid GeneExpert

Are there many FDA-approved molecular diagnostic tests?

Advantage of FDA Approved Tests

If you use FDA approved procedures without modification, you can be inspected under the CAP Microbiology Checklist.

If you make procedural or specimen changes or use non-FDA approved procedures, you must use the more extensive Molecular Pathology Checklists.

FDA Approved ID Tests

MRSA Surveillance (GeneXpert, BD/Geneohm SmartCycler)

Chlamydia trachomatis Neisseria gonorrhoeae Gardnerella, Trichomonas

vaginalis and Candida spp. Cytomegalovirus (qual) HCV qual/quant HIV quant

Legionella pneumophila Group A strep Group B strep (GeneXpert,

SmartCycler) Mycobacterium tuberculosis Mycobacterial speciation HPV HIV drug resistance Enterovirus (GeneXpert)

www.amp.org/ click on Resources then FDA Approved Tests

What other types of tests are available?

In-house developed (home brew) tests using ASR and RUO reagents

Analyte Specific Reagent (ASR)

ASRs are raw (key analyte-specific) materials and components used to develop laboratory assays.

ASR manufacturers are not permitted to make any claims regarding analytical or clinical performance of the ASR.

ASR manufacturers are not permitted to provide information on assay methods or techniques.

http://www.devicelink.com/mddi/archive/03/02/018.html

Major ASR and RUO Suppliers

Abbott Molecular Systems Artus GMBH / Qiagen Cepheid EraGen Roche Molecular Systems

Artus GMBH Cytomegalovirus Dengue Virus EBV Enterovirus Hepatitis A Hepatitis B HSV 1, 2 Influenza Parvovirus B19 SARS-Coronavirus Varicella Zoster Virus West Nile Virus

Bacillus anthracis Borrelia Chlamydia trachomatis Campylobacter L. monocytogenes Mycobacterial differentiation M. tuberculosis Salmonella E. histolytica Malaria

Abbott/Celera ViroSeq™ HIV Genotyping System* HIV qual/quant* Hepatitis C Virus qual/quant Hepatitis C Virus genotyping Varicella zoster virus Epstein-Barr Virus Cytomegalovirus Hepatitis B virus qual/quant

*FDA approved product

Roche Molecular Systems

COBAS TaqMan HBV COBAS TaqMan HCV COBAS Taqman HIV*

Bordetella IS481/1001 CMV UL54 EBV Enterococcus HSV 1/2, MRSA Pseudomonas Staphylococcus Strep A, Strep B pts1 gene VRE VZV

*FDA-approved product

Mayo Procedures for LightCycler

EBV quantitation Cytomegalovirus quantitation Herpes Simplex Virus Varicella Zoster virus Bordetella pertussis

Group A Strep Group B Strep VRE Mec A

Cepheid ASR Program Current ASR Products

Bordetella pertussis HSV Typing & Non-Typing Enterovirus* Parvo B19 Staph Protein A Mec A* B. pertussis/parapertussis Mycoplasma pneumoniae Flu A/Flu B RSV Norovirus Group B Strep*

Coming Soon

C. pneumoniae Legionella VanA/VanB Enterococcus

*FDA-approved products

Other Equipment Needed

Laminar airflow hoods/dead air boxes -20oC and -70oC freezers Centrifuges, Microfuges Thermocyclers/heat blocks/water baths? Plate washer/plate reader/fluorimeter? UV lights?