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:
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Detection technologies
Commercial detection kits
Analyte specific reagents
Instrumentation options
Growth Projections
Test Category
$ Millions (2001)
Est. Growth/Year
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%
Coagulation
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
Detection
Reagent
Prep
End-point
PCR
Sample
Prep
Analysis
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?
PCR Amplification Plot
Plateau
Linear
Phase
Exponential
Phase
Threshold
CT
Baseline
Cycle Number
Dilution Series
Relative fluorescence
2.3
1-108 virus copies
2.1
1.9
1.7
1.5
1.3
1.1
0.9
1
5
9
13
17
21
25
29
33
37
Cycle number
41
45
49
53
57
61
TaqMan RT-PCR Results
For Enterovirus
CSF
Urine
Serum
+ Control
Cycle Number
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?
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10% bleach (freshly made)
Ultraviolet light
Nucleolytic agents
Some acids
Contamination Control
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Engineering controls
Procedural controls
Chemical controls within the assay
Surveillance
Engineering Controls
Work flow
Reagent
Preparation
Specimen
Preparation
Reaction
Setup and
Amplification
Dedicated Hallway
Product
Analysis
Engineering Controls - Kits
Work flow
Reagent
Preparation
Specimen Preparation
Amplification Setup
Amplification,
Detection, and
Product Analysis
Real-Time PCR
Work flow
Reagent
Preparation
Specimen Preparation
Amplification Setup in
Biosafety Cabinet
Amplification,
Detection, and
Product Analysis
Disposables/Procedural Controls
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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?
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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?
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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 Technology
A
B
Lysis
C
Bind
D
Wash
Elute
Qiagen Spin Columns
 Silica membrane plus
chaotropic salts
 No organic extraction
 No ethanol precipitation
 DNA or RNA, Both
 Wide range of clinical samples
 Mini, midi, and maxi columns
QIAcube Spin-Column Processing
Commercial Spin Columns
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Qiagen
Clontech
Amresco
Gentra Systems
Stratagene
Roche Molecular
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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 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 EZ1
 Fully automated
 Pre-programmed protocol
cards
 Prefilled, sealed reagent
cartridges
 Small footprint
 1-6 samples in 15-30 min
 200 to 350 l of sample
 Variety of samples
Qiagen BioRobot EZ1
MagNA 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
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Abbott Molecular Systems
Artus GMBH / Qiagen
Cepheid
EraGen
Roche Molecular Systems
Artus GMBH
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Cytomegalovirus
Dengue Virus
EBV
Enterovirus
Hepatitis A
Hepatitis B
HSV 1, 2
Influenza
Parvovirus B19
SARS-Coronavirus
Varicella Zoster Virus
West Nile Virus
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Bacillus anthracis
Borrelia
Chlamydia trachomatis
Campylobacter
L. monocytogenes
Mycobacterial differentiation
M. tuberculosis
Salmonella
E. histolytica
Malaria
Abbott/Celera
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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
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COBAS TaqMan HBV
COBAS TaqMan HCV
COBAS Taqman HIV*
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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
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EBV quantitation
Cytomegalovirus quantitation
Herpes Simplex Virus
Varicella Zoster virus
Bordetella pertussis
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Group A Strep
Group B Strep
VRE
Mec A
Current ASR Products
Coming Soon
Cepheid
ASR
Program
 C. pneumoniae
 Bordetella pertussis
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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*
 Legionella
 VanA/VanB
 Enterococcus
*FDA-approved products
Other Equipment Needed
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Laminar airflow hoods/dead air boxes
-20oC and -70oC freezers
Centrifuges, Microfuges
Thermocyclers/heat blocks/water baths?
Plate washer/plate reader/fluorimeter?
UV lights?