the application of real-time pcr in the diagnosis of infectious disease the application of real-time...
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The Application of Real-Time PCR
in the Diagnosis of Infectious Disease
The Application of Real-Time PCR
in the Diagnosis of Infectious Disease
T.P.Sloots Clinical Virology Research Unit, RCH, & Microbiology, QHPS.
Why should we use PCR?Why should we use PCR?
• Very sensitive (1 copy – 10 copies of DNA)
• Can detect organisms that cannot be isolated
• Rapid (TAT = < 24 hrs)
• Very sensitive (1 copy – 10 copies of DNA)
• Can detect organisms that cannot be isolated
• Rapid (TAT = < 24 hrs)
Disadvantages of PCRDisadvantages of PCR
• Technically demanding
• Can be expensive
• Risk of contamination
• Need rigid QC
• Technically demanding
• Can be expensive
• Risk of contamination
• Need rigid QC
PCRPCRPCRPCR
LightCyclerRoche
real-timereal-time real-time
real-time PCRreal-time PCR
iCyclerBioRad
7700Applied Biosystems
5700Applied Biosystems
FluorTrackerStratagene
FluorImagerMolecular Dynamics
hardwarehardware
real-timereal-time real-time
real-timereal-time
integrated systemamplifies & detects
constant monitoringfluorescent probes
rapid cycling times
quantitative
low contamination
riskassay design
PCRPCR
fast turn-around sealed system
• Microtitre plate format, sealed system
• Processes 96 samples in 2½ hours
• Real-time - amplification and detection
• Quantitative results
• Uses a fluorogenic probe, with reporter & quencher dyes
• Taq DNA polymerase has 5’-3’ exonuclease activity
ABI 7700
real-timereal-time real-time
TaqManTaqManhardwarehardware
ABI Biosystems
real-timereal-time
real-timereal-timeTaqManTaqMan
real-time
Amplicon
FRET
Amplicon
Emission
EXTENSION
ANNEALING
Excitation
5’-3’ exonuclease
Reporter
Quencher
• Real-time detection
• Quantitative results
• Hybridization probes
• Can detect 2 targets simultaneously
• Uses capillaries (10-20ul)
• 32 samples / 60 minutes
• Sealed system – contamination free
• Real-time detection
• Quantitative results
• Hybridization probes
• Can detect 2 targets simultaneously
• Uses capillaries (10-20ul)
• 32 samples / 60 minutes
• Sealed system – contamination free
1.
2.
3.
4.
FRET (Fluorescence Resonance Energy Transfer) FRET (Fluorescence Resonance Energy Transfer)
using adjacent hybridization probes using adjacent hybridization probes
FRET (Fluorescence Resonance Energy Transfer) FRET (Fluorescence Resonance Energy Transfer)
using adjacent hybridization probes using adjacent hybridization probes
FITC
Red 640
P Phosphate
FRET Emission
P
Excitation
Amplicon
real-timereal-timereal-time
LightCyclerLightCyclerOperation
DenaturationDenaturation
95oC
FRET Emission
P
ExcitationTm
55oC
Primer/Probe AnnealingPrimer/Probe Annealing
FluorimeterReading
FluorimeterReading
72oC
Primer ExtensionPrimer Extension
• Detection of Infectious Disease agents
• Target Characterisation
• Determining Microbial Load (quantitation)
• Detection of Infectious Disease agents
• Target Characterisation
• Determining Microbial Load (quantitation)
62 (23%) were culture positive, confirmed by antigen detection with MoAb (27= HSV-1, 35= HSV-2).
113 (42%) were LC-PCR positive following extraction of VTM using a glass fibre column (Qiagen).
51 were LC-PCR positive and culture negative. All these were confirmed as HSV by sequencing.
1 culture + / PCR - specimen. Was negative by repeat culture, and remained negative by “in house” PCR using different primers
266 swabs from multiple sites were collected in VTM for HSV culture.
Characterisation of HSV by melting curve Characterisation of HSV by melting curve
DNA pol
HSV
HSV-1
HSV-2
mismatch
Hybridisation probes (to HSV-1)
no mismatch
Amplicon
Primers commonto HSV 1 & 2
HSV 2 HSV 1
Melting Curve Analysis
HSV 1
HSV 2
55oC
HSV 1
HSV 2
73oC
HSV 1
HSV 2
67oC
Microbial load testingMicrobial load testing
• For commensal organisms determine a “normal” microbial load. Elevated level determines infection.
• Detect active infection by increasing load
• Detect anti-viral drug resistance (CMV, HSV)
0.5
1.5
2.5
0 10 20 30 40 50 60 70
Cycles
F2/F
1
NEG
10
100
1000
10000
100000
1000000
Threshold Cycle
Microbial Load Testing
0
10
20
30
40
50
1 2 3 4 5 6
Concentration log 10
Th
resh
old
Cycle
0.5
1.5
2.5
0 10 20 30 40 50 60 70
Cycles
F2/F
1
Test Sample
Threshold
Threshold Cycle
Threshold Cycle = 35Load = 103.8 copies/ml
PRACTICAL APPLICATIONPRACTICAL APPLICATION
Monitoring CMV disease in transplant patients, particularly Bone Marrow Transplant recipients.
1. Early detection of disease progression to apply appropriate drug therapy
2. Detect ganciclovir drug resistance
0
1000
2000
3000
4000
5000
6000
7000
8000
Sampling Time (Wks)
40
30
20
10
0
AntigenemiaPositive cells
per 200,000 cells
AntigenemiaPositive cells
per 200,000 cells
g
eno
me
cop
ies
q-PCR
1 2 3 4 5 6 7 8 9 10 11
Ganciclovir
BMT PATIENT 1BMT PATIENT 11 2 3 4 5 6 7 8 9 10 11
ROCHE PCR
“in house” PCR
Antigenemia
0
1000
2000
3000
4000
5000
6000
7000
8000
q-PCR
1 2 3 4 5 6 7 8 9 10 11 12 13
80
60
40
20
0
Ganciclovir
Foscarnet
BMT PATIENT 2BMT PATIENT 2
Sampling Time (Wks)
AntigenemiaPositive cells
per 200,000 cells
AntigenemiaPositive cells
per 200,000 cells
g
eno
me
cop
ies
1 2 3 4 5 6 7 8 9 10 11 12 13
ROCHE PCR
“in house” PCR
DISADVANTAGES OF REAL-TIME PCRDISADVANTAGES OF REAL-TIME PCR
Current technology has limited capacity for multiplexing. Simultaneous detection of 2 targets is the limit.
Development of protocols needs high level of technical skill and/or support. (Requires R&D capacity and capital)
High capital equipment costs ($ 50,000 -160,000).
ADVANTAGES OF REAL-TIME PCRADVANTAGES OF REAL-TIME PCR
Rapid cycling times (1 hour)
High sample throughput (~200 samples/day)
Low contamination risk (sealed reactions)
Very sensitive (3pg or 1 genome eq of DNA)
Broad dynamic range (10 - 1010 copies)
Reproducible (CV < 2.0 %)
Allows for quantitation of results
Software driven operation
No more expensive than “in house” PCR ($15/test)
PCR DetectionPCR Detection
• TaqMan and LC utilse probes
• Non-specific reactions with probe may occur
• Number of chromophors is limited
• Alternative detection technologies
- molecular beacons
- multiple arrays (gene chip)
Alternative Detection
Technology
Alternative Detection
Technology
Molecular BeaconsMolecular Beacons
• Hairpin shaped hybridisation probes
• Contain fluorophor and quencher
• Added to PCR reaction mix
• Hybridise to target during PCR
• Monitor end-point PCR
• Real-time PCR monitoring
• Allows more flexible thermocycling parameters
Amplicon
molecular beaconsmolecular beacons
A
B
C
FRET
real-timereal-timereal-time
real-timereal-time
Reporter
Non-fluorescent Quencher
Excitation
ANNEALING
Molecular BeaconsMolecular Beacons
APPLICATIONSAPPLICATIONS
• Detection of amplification products (real time, end-point)
• Multicolour beacons detect multiple targets (8)
• Better detection of single point mutation
• Drug resistance analysis
• Non-PCR hybridisation analysis (in situ labeling)
Multiple DNA ArraysMultiple DNA Arrays
• Detection of thousands of gene sequences simultaneously
• Capacity for minitiarisation
• Suitable for automation
• Enormous analytical power
• Detection of thousands of gene sequences simultaneously
• Capacity for minitiarisation
• Suitable for automation
• Enormous analytical power
Multiple DNA ArraysMultiple DNA Arrays
Use of Multiple Arrays involves 5 steps
• Preparation of array containing capture probes
• Isolation, purification and labeling of test sample DNA
• Hybridisation of test sample DNA to capture array
• Detection of captured DNA hybrids
• Data analysis
Genechip ArrayImmobilised capture probes
Labeledsample DNA
x
x
x
x
Conjugatedfluorophor
Image of array
pre (red)/post
(green)
1000’s genes/pcr amplified
segments
robot loaded glass
slide
microarrays (<200um)microarrays (<200um)
share data
good controls
gene arraysgene arrays
gene arraysgene arraysgene arrays
known grid positions
hybridise 2 fluor-tag samples
illuminatedconfocal microscope
quantitation
interpretation
Microarrays (Gene Chips)Microarrays (Gene Chips)
APPLICATIONS
• Genome mutational analysis• Multiple drug resistance• Monitor gene expression in cells• Pharmocogenomics• Screening for multiple infectious
agents