the application of real-time pcr in the diagnosis of...
TRANSCRIPT
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
real-timereal-time real-timerealreal--timetime
integrated systemamplifies & detects
constant monitoringfluorescent probes
rapid cycling times
quantitative
low contamination risk
assay 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-timerealreal--timetime
TaqManTaqMan
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
real-timereal-time real-time
LightCyclerLightCyclerHardware
FRET (Fluorescence Resonance Energy Transfer)
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-time real-time
LightCyclerLightCyclerFRETFRET
FRET Emission
P
ExcitationTm
55oC
Primer/Probe AnnealingPrimer/Probe Annealing
FluorimeterReading
FluorimeterReading
• Detection of Infectious Disease agents
• Target Characterisation
• Determining Microbial Load (quantitation)
• Detection of Infectious Disease agents
• Target Characterisation
• Determining Microbial Load (quantitation)
real-timereal-time real-time
LightCyclerLightCyclerApplications
Routine Use
HSV-1 & HSV-2JCV & BKVPneumocystis cariniiNeisseria meningitidisNeisseria gonorrhoeaVZVToxoplasma gondiiBordetella pertussis
Validation
Influenza ARSV
CMV (quantitative)
Under Development
Enterovirus (group)16S Bacterial rRNAFlu/RSV multiplex
real-timereal-time real-time
LightCyclerLightCyclerApplications
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
real-timereal-time real-time
LightCyclerLightCyclerHSV PCR
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 mismatchAmplicon
real-timereal-time real-time
LightCyclerLightCyclerApplication
Primers commonto HSV 1 & 2
real-timereal-timereal-timerealreal--timetime
PCR quantitationPCR quantitation
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
/F1
NEG
10
100
1000
10000
100000
1000000
real-timereal-timereal-timerealreal--timetime
PCR quantitationPCR quantitation
Threshold Cycle
Microbial Load Testing
0
10
20
30
40
50
1 2 3 4 5 6
Concentration log 10
Th
resh
old
Cycl
e
0.5
1.5
2.5
0 10 20 30 40 50 60 70
Cycles
F2
/F1
Test Sample
Threshold
Threshold Cycle
Threshold Cycle = 35Load = 103.8 copies/ml
PRACTICAL APPLICATIONPRACTICAL APPLICATION
real-timereal-timereal-timerealreal--timetime
PCR quantitationPCR quantitation
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
geno
me
copi
es
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
real-timereal-time real-time
realreal--time PCRtime PCRViral LoadViral Load
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
geno
me
copi
es
1 2 3 4 5 6 7 8 9 10 11 12 13
ROCHE PCR
“in house” PCR
real-timereal-timerealreal--time PCRtime PCR
Viral LoadViral Load
real-timereal-timerealreal--time PCRtime PCR
SummarySummary
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 resultsSoftware driven operationNo more expensive than “in house” PCR ($15/test)
real-timereal-timerealreal--time PCRtime PCR
Summary Summary
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)
Amplicon
molecular beaconsmolecular beacons
A
B
C
FRET
real-timereal-time real-timerealreal--timetime
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)