Molecular Testing and Clinical Diagnosis

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Molecular Testing and Clinical Diagnosis

• Amplified nucleic acid testing
• Part III

2
Objectives At the end of this lesson the
student will

• Describe and evaluate types of target sequences
  (DNA, mRNA, tRNA, rRNA) (C3)
• Describe and compare amplification processes
  including (C3)
• Basic steps of an amplification process
• Principles of methods available
• PCR, LCR, SDA, NASBA, TMA
• List and describe the function PCR components in
  the reaction mix (C2)
• Describe the variations of PCR process (C2)
• LCR
• Reverse Transcription-PCR
• Real time PCR

3
Objectives At the end of this lesson the
student will

• Explain the application of PCR to STR testing
  (C2)
• Paternity testing
• Forensic testing
• RFLP mapping
• Describe the significance of the following PCR
  considerations (C2, A2)
• Contamination
• Quality control
• Lab space allocation

4
Strand Displacement Amplification- BDPobeTec ET
system

• One hour assay
• fluorescence detection
• automated and semi-automated systems
• pre-dispensed reagent devices

5
Strand Displacement Process

• step 1 primer hybridization
• step 2 primer extensions by DNA polymerase
  leads to strand displacement
• step 3 extended probe binds complimentary strand
• step 4 probe is extended creating BsoBI site
• step 5 BsoBI cleaves dsDNA

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Detection linked with amplification.
Target must be amplified and double stranded to
enable the restriction enzyme to function.
Fluorescence only occurs when there is cleavage.
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SDA lends itself to automation since it is
isothermal.
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Transcription-Mediated Amplification

• RNA transcription amplification system using two
  enzymes RNA polymerase and reverse transcriptase
• Isothermal amplification of nucleic acid target
  producing RNA product amplification
• Rapid kinetics results in excess of ten
  billion-fold amplification within 15-30 minutes
• Combined with Hybridization Protection Assay
  detection in a single tube format

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Transcription-Mediated Amplification Components

• Primers Oligonucleotides that hybridize to
  target and initiate the reaction
• Nucleotides
• Enzymes drive the reaction
• T7 RNA polymerase
• transcribes RNA from DNA
• Reverse transcriptase (MLV)
• synthesizes DNA from RNA or DNA
• RNAse H activity degrades RNA after it has been
  copied into DNA

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Transcription-Mediated Amplification
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TMA Gen-Probe Second GenerationAPTIMATM Assays

• Target Capture sample processing partially
  purifies target nucleic acid
• Transcription-Mediated Amplification-- amplified
  target
• Dual Kinetic Assay (DKA) technology
  simultaneously detects two organisms

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Gen-Probe Proprietary Target Capture Technology
N
Bead
TTTTTTTTTTTTTT
Oligo
TTTTTTTTTTTTTT
Capture
Oligo "Tail"
one
micron
Magnet
AAAAAAAAAAAAAAAAAA
magnetic

TTTTTTTTTTTTTT
particle
TTTTTTTTTTTTTT
.....GAUCGAUCCCCCCUAGCGGUGCAUCUAGCAUCUA....
TTTTTTTTTTTTTT
GGATCGCCACGTAGATCGGCCTC
TTTTTTTTTTTTTT

TTTTTTTTTTTTTT
S
Capture
Sequence
These are washed away
Cell debris
Protein
Target
Sequence
non-specific
Plasma
DNA/RNA
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Detection by Dual Kinetic Assay (DKA) Technology

• Hybridization Protection Assay (HPA) Technology
• Two modified acridinium
• ester labels with different
• light-off kinetics on
• different DNA probes
• Flasher fast
• Glower slow
• Simultaneous detection of different organisms

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Dual Kinetic Assay (DKA)
120,000
CT GC
CT
100,000
GC
80,000
RLU
60,000
40,000
20,000
0
.52
.68
.84
1.00
1.16
1.32
1.48
1.64
1.80
1.96
.04
.20
.36
Time in Seconds
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TMA Amplicon Production
TMA Amplicon Production
Starting with 1000 copies of Target
1E14
1E12
1E10
1E8
Amplicon copies
1E6
1E4
1E2
1E0
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (min.)
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Gen-Probe Instrumentation Systems

• Fully automated, APTIMATM amplification assays
  for TIGRISTM
• Target Capture system
• VIDAS dual platform Amplified assays and
  immunoassays

from bioMérieux
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Challenges with Current Nucleic Acid
Amplification Tests

• Carry-over contamination can cause false
  positives
• Verification of positive results is difficult
• Inhibition can cause false negatives
• Compared with current microbiology tests
• Increased labor
• Higher cost
• Low throughput

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Methods for Control of Carryover Contamination in
automated TMA Assays

• Unidirectional workflow
• Single-tube format
• Oil as a barrier to the environment
• HPA format eliminates wash steps and potential
  aerosols
• Treatment of RNA amplicon with detection reagents
• Bleach destroys nucleic acids

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Comparison of TMA with PCR and LCR Amplification
Methods
TMA (
Gen-Probe )
PCR (
Roche )
LCR ( Abbott )
Ligase DNA polymerase
RNA polymerase Reverse transcription
Enzymes
DNA polymerase
Thermal
Isothermal reaction
Thermal cycling
Thermal cycling
Conditions
Amplified
RNA
DNA
DNA
Product
Homogeneous
Wash step
assay no wash
Wash step
required
Wash step required
steps
Detection System
Chemiluminescence
Absorbance
Fluorescence
Special
Thermal cycler,
equipment
microtiter plate
Thermal cycler,
Luminometer
needed
reader/washer
LCx instrument
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Gen-Probe HIV-1/HCV dual-assay protocol for blood
supply



Step One SampleProcessing Extract RNA 90
minutes (Hybridized target captured on to
microparticles)
Step Two TMA Add Amplification Reagent, Oil
Reagent 10 minutes 41.5C Add Reverse
Transcriptase, RNA Polymerase 60 minutes 41.5C
Step Three HPA Add Probe Reagent(Hybridizes to
amplicon) 15 minutes 60C Add Selection
Reagent 10 minutes 60C Read in Luminometer

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Pooling Scheme
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128 Pool
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128 Donations
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Resolution Testing
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128
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1
1
Reactive Pool
Identification of Single Donation
Test Primary Pools
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HCV Panel 6211 Virologic/Serologic Profile
46 Days
S/CO
HCV PCR Quantitation
Days
? PCR
E991685 7-14-99 25
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Summary Amplification Methods

• Much like a culture technique, they increase
  likelihood of detection and identification
• Enzymes are used to increase target sequence for
  detection
• May be automated or semi-automated more easily if
  isothermal

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Summary Amplification Methods

• Increased sensitivity
• amplification
• detection systems
• Specificity
• primers
• probe/detection systems