The Application of Real-Time PCR

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The Application of Real-Time PCR in the Diagnosis
of Infectious Disease
T.P.Sloots Clinical Virology Research
Unit, RCH, Microbiology, QHPS.
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PCR
PCR
PCR
Why should we use PCR?

• Very sensitive (1 copy 10 copies of DNA)
• Can detect organisms that cannot be isolated
• Rapid (TAT lt 24 hrs)

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real-time
real-time
real-time PCR
real-time
hardware
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real-time
real-time
real-time
real-time
PCR
amplifies detects
integrated system
constant monitoring
fluorescent probes
rapid cycling times
fast turn-around
low contamination risk
sealed system
assay design
quantitative
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real-time
real-time
TaqMan
real-time
hardware
ABI Biosystems
ABI 7700

• 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

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real-time
real-time
real-time
real-time
TaqMan
Emission
Excitation
FRET
Amplicon
ANNEALING
EXTENSION
Amplicon
5-3 exonuclease
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real-time
real-time
LightCycler
real-time
Hardware

• Real-time detection
• Quantitative results
• Hybridization probes
• Can detect 2 targets simultaneously
• Uses capillaries (10-20ul)
• 32 samples / 60 minutes
• Sealed system contamination free

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real-time
real-time
LightCycler
real-time
Set Up
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real-time
real-time
LightCycler
real-time
Hardware
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real-time
real-time
LightCycler
real-time
FRET
FRET (Fluorescence Resonance Energy Transfer)
using adjacent hybridization probes

Red 640
Phosphate

Emission
Amplicon
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real-time
real-time
LightCycler
real-time
Operation
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95oC
Denaturation
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Emission
Tm
55oC
Primer/Probe Annealing
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72oC
Primer Extension
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real-time
real-time
LightCycler
real-time
Applications

• Detection of Infectious Disease agents
• Target Characterisation
• Determining Microbial Load (quantitation)

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real-time
real-time
LightCycler
real-time
HSV PCR
266 swabs from multiple sites were collected in
VTM for HSV culture.

• 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

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real-time
real-time
LightCycler
real-time
Characterisation of HSV by melting curve
Application
HSV
DNA pol
Primers common to HSV 1 2
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HSV 1
HSV 2
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real-time
real-time
real-time
real-time
PCR quantitation
Microbial 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)

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real-time
real-time
real-time
real-time
PCR quantitation
Microbial Load Testing
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Threshold Cycle
Concentration log 10
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real-time
real-time
real-time
real-time
PCR quantitation
PRACTICAL APPLICATION
Monitoring CMV disease in transplant patients,
particularly Bone Marrow Transplant recipients.

• Early detection of disease progression to apply
  appropriate drug therapy
• Detect ganciclovir drug resistance

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real-time
real-time
real-time PCR
real-time
Viral Load
BMT PATIENT 1
40 30 20 10 0
Ganciclovir
q-PCR
Antigenemia Positive cells per 200,000 cells
genome copies
Antigenemia
1 2 3 4 5 6
7 8 9 10 11
Sampling Time (Wks)
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real-time
real-time PCR
real-time
Viral Load
BMT PATIENT 2
1 2 3 4 5 6 7
8 9 10 11 12 13
ROCHE PCR
in house PCR
80 60 40 20 0
Ganciclovir
Foscarnet
Antigenemia Positive cells per 200,000 cells
genome copies
q-PCR
1 2 3 4 5 6 7
8 9 10 11 12 13
Sampling Time (Wks)
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real-time
real-time PCR
real-time
Summary
DISADVANTAGES 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 RD
  capacity and capital)
• High capital equipment costs ( 50,000 -160,000).

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real-time
real-time PCR
real-time
Summary
ADVANTAGES 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 lt 2.0 )
• Allows for quantitation of results
• Software driven operation
• No more expensive than in house PCR (15/test)

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PCR 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)

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Alternative Detection Technology
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Molecular 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
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real-time
real-time
real-time
real-time
molecular beacons

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Molecular Beacons
APPLICATIONS

• 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)

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Multiple DNA Arrays

• Detection of thousands of gene sequences
  simultaneously
• Capacity for minitiarisation
• Suitable for automation
• Enormous analytical power

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Multiple 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

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gene arrays
gene arrays
gene arrays
gene arrays
microarrays (lt200um)
1000s genes/pcr amplified segments
pre (red)/post (green)
good controls
share data
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Microarrays (Gene Chips)

• APPLICATIONS
• Genome mutational analysis
• Multiple drug resistance
• Monitor gene expression in cells
• Pharmocogenomics
• Screening for multiple infectious agents