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