molecular testing and clinical diagnosis
DESCRIPTION
Molecular Testing and Clinical Diagnosis. Amplified nucleic acid testing Part III. 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) - PowerPoint PPT PresentationTRANSCRIPT
Molecular Testing and Clinical Diagnosis
Amplified nucleic acid testing
Part III
• 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
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
Objectives: At the end of this lesson the student will:
Strand Displacement Amplification- BDPobeTec ET system• One hour assay
• fluorescence detection
• automated and semi-automated systems
• pre-dispensed reagent devices
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
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.
SDA lends itself to automation since it is isothermal.
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
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
Transcription-Mediated Amplification
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
.....GAUCGAUCCCCCCUAGCGGUGCAUCUAGCAUCUA....
•••••••••••••••••••••••••••••••••••••
one micron
magnetic particle
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
––TTTTTTTTTTTTTT
AAAAAAAAAAAAAAAAAAGGATCGCCACGTAGATCGGCCTC
Mag
net
N
S
Bead Bead OligoOligo
Capture Capture Oligo "Tail"Oligo "Tail"
Target Target SequenceSequence
Capture Capture SequenceSequenceThese are washed away:These are washed away:
non-specific non-specific DNA/RNADNA/RNA
ProteinProteinCell Cell debrisdebris
PlasmaPlasma
••••••••••••••••••••••••••
Gen-Probe Proprietary Target Capture Technology
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
Dual Kinetic Assay (DKA)
0
20,000
40,000
60,000
80,000
100,000
120,000
.04 .20 .36 .52 .68 .84 1.00 1.16 1.32 1.48 1.64 1.80 1.96
CT + GC
CT
GC
RL
U
Time in Seconds
1E0
1E2
1E4
1E6
1E8
1E10
1E12
1E14
Am
plic
on c
opie
s
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min.)
TMA Amplicon ProductionStarting with 1000 copies of Target
TMA Amplicon Production
Gen-Probe Instrumentation Systems
Fully automated, APTIMATM amplification assays for TIGRISTM
Target Capture system
VIDAS* dual platform: Amplified assays and immunoassays
*from bioMérieux
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
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
Comparison of TMA with PCR and LCR Amplification Methods
TMA (Gen-Probe ) PCR (Roche ) LCR ( Abbott )
Thermal
Conditions Isothermal reaction Thermal cycling Thermal cycling
AmplifiedProduct RNA DNA DNA
Wash step
Homogeneousassay no washsteps Wash step required
Wash steprequired
Detection System Chemiluminescence Absorbance Fluorescence
Specialequipmentneeded Luminometer
Thermal cycler,microtiter plate
reader/washerThermal cycler,LCx instrument
RNA polymeraseReverse transcription DNA polymerase
LigaseDNA polymeraseEnzymes
Step OneStep One
SampleSampleProcessingProcessing
Extract RNAExtract RNA
~ 90 minutes~ 90 minutes
(Hybridized target (Hybridized target
captured on to captured on to microparticles)microparticles)
Gen-Probe HIV-1/HCV dual-assay protocol for blood supply
Step TwoStep Two
TMATMA Add Add
Amplification Amplification
Reagent, Oil Reagent, Oil ReagentReagent
10 minutes 10 minutes 41.5°C41.5°C
Add Reverse Add Reverse Transcriptase, Transcriptase,
RNA PolymeraseRNA Polymerase
60 minutes 60 minutes 41.5°C41.5°C
Step ThreeStep Three
HPAHPA Add Probe Add Probe
ReagentReagent(Hybridizes to (Hybridizes to
amplicon)amplicon)
15 minutes 60°C15 minutes 60°C
Add Selection Add Selection ReagentReagent
10 minutes 60°C10 minutes 60°C
Read in Read in LuminometerLuminometer
Pooling Scheme
128Donations
16 16
16 16
16 16
16 16
128Pool
11
11
11
1
Resolution Testing
Identification of SingleDonation
16 16
16 16
16 16
16 16
ReactivePool
11
11
11
11 1
Test Primary Pools
128
HCV Panel 6211 – Virologic/Serologic Profile
DaysDays
S/C
OS
/CO
HC
V P
CR
Qua
ntita
tion
HC
V P
CR
Qua
ntita
tion
46 Days46 Days
PCR
100
1,000
10,000
100,000
1,000,000
10,000,000
0 20 40 60 80 100 120 140 160 180 200
0
1
2
3
4
5
6
7
8
9
10
Antibody
E991685 7-14-99 25
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
Summary Amplification Methods
• Increased sensitivity– amplification– detection systems
• Specificity – primers– probe/detection systems