molecular diagnostics – how to get started
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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Molecular Diagnostics – How To Get Started
Danny L. Wiedbrauk, Ph.D.
Warde Medical Laboratory
Ann Arbor, Michigan
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Molecular Diagnostics
Fastest growing area in laboratory medicine.
Increasing numbers of: Detection technologies Commercial detection kits Analyte specific reagents Instrumentation options
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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
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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.
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Traditional PCR Methods
ReagentPrep
SamplePrep
End-pointPCR
Analysis
Detection
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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
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Real-Time PCR –The Enabling Technology
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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
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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
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What is Real-Time PCR?
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Cycle Number
PCR Amplification PlotF
luor
esce
nce
(R
n)
Threshold CT
Plateau
Baseline
Exponential Phase
Linear Phase
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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
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+ Control
Serum
Urine
CSF
Cycle Number
TaqMan RT-PCR ResultsFor Enterovirus
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What This Means to You
These new technologies allow general laboratories such as Microbiology and Hematology to do nucleic acid testing.
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How do we get started?
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Contamination Control
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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.
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Contamination Control
DNA is very hardy and is resistant to many traditional decontamination procedures including: Alcohols and organic solvents Detergents and disinfectants Autoclaving Drying
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What Works?
10% bleach (freshly made) Ultraviolet light Nucleolytic agents Some acids
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Contamination Control
Engineering controls Procedural controls Chemical controls within the assay Surveillance
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Engineering Controls
Reagent Preparation
Specimen Preparation
Reaction Setup andAmplification
Product Analysis
Dedicated Hallway
Work flow
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Engineering Controls - Kits
Reagent Preparation
Specimen PreparationAmplification Setup
Amplification,Detection, and Product Analysis
Work flow
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Real-Time PCR
Reagent Preparation
Specimen PreparationAmplification Setup inBiosafety Cabinet
Amplification,Detection, and Product Analysis
Work flow
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Disposables/Procedural Controls
Aerosol resistant tips Separate lab coats/gloves Dedicated equipment Unidirectional workflow Only one tube open Low level positive controls
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Procedural Controls
Dedicated equipment Separate lab
coats/gloves
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Surveillance
Wipe testing Monitoring negative controls Monitoring prevalence rates
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Question
How can you do wipe testing in a lab that routinely grows the infectious agent?
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Wipe Testing
Wipe testing can reinforce the need to disinfect the environment daily.
Can improve infection control in lab
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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
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What Will You Be Doing?
Extracting nucleic acids Some type of amplification technology Signal or nucleic acid detection Interpretation of results
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Extraction Systems
Remove inhibitory and interfering substances without significantly altering the amount or quality of the target nucleic acid
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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
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Spin Column TechnologySpin Column Technology
LysisLysis BindBind WashWash EluteElute
AA BB CC DD
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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
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QIAcube Spin-Column Processing
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Commercial Spin Columns
Qiagen Clontech Amresco Gentra Systems Stratagene Roche Molecular
QIAmp, Rneasy Nucleospin Cyclo-Prep Generation Capture Strata Prep High Pure
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Magnetic Particle Technology
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Magnetic Particle Kits
Cortex Biochem Promega Dynal
Roche Molecular
MegaZorb
MagnaSil
Dynabeads
DNA Direct
DNA Isolation Kit
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Magnetic Separators
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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
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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
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Qiagen BioRobot EZ1Qiagen BioRobot EZ1
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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
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Major Clinical Systems for Real-Time PCR
ABI 7300/7500
Roche LightCycler
Cepheid SmartCycler Cepheid GeneExpert
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Are there many FDA-approved molecular diagnostic tests?
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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.
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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
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What other types of tests are available?
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In-house developed (home brew) tests using ASR and RUO reagents
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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
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Major ASR and RUO Suppliers
Abbott Molecular Systems Artus GMBH / Qiagen Cepheid EraGen Roche Molecular Systems
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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
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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
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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
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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
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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
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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?