1. 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.

2. 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 PCR Technically demanding Can be expensive Risk of contamination Need rigid QC Technically demanding Can be expensive Risk of contamination Need rigid QC PCR PCR

3. LightCycler Roche real-time real-time PCR iCycler BioRad 7700 Applied Biosystems 5700 Applied Biosystems FluorTracker Stratagene FluorImager Molecular Dynamics hardware

4. real-time real-time integrated system amplifies & detects constant monitoring fluorescent probes rapid cycling times quantitative low contamination risk assay design PCR fast turn-around sealed system

5. 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-time TaqMan hardware ABI Biosystems

6. real-time real-time TaqMan Amplicon FRET Amplicon Emission EXTENSION ANNEALING Excitation 5’-3’ exonuclease Reporter Quencher

7. 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

8. 1. 2. 3. 4.

10. FRET (Fluorescence Resonance Energy Transfer) using adjacent hybridization probes using adjacent hybridization probes FRET (Fluorescence Resonance Energy Transfer) using adjacent hybridization probes using adjacent hybridization probes FITC Red 640 P Phosphate FRET Emission P Excitation Amplicon

11. real-time LightCycler Operation

12. Denaturation 95 o C

13. FRET Emission P Excitation TmTm 55 o C Primer/Probe Annealing Fluorimeter Reading Fluorimeter Reading

15. 72 o C Primer Extension

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

17.  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.

18. Characterisation of HSV by melting curve DNA pol HSV HSV-1 HSV-2 mismatch Hybridisation probes (to HSV-1) no mismatch Amplicon Primers common to HSV 1 & 2

19. HSV 2 HSV 1 Melting Curve Analysis HSV 1 HSV 2 55 o C HSV 1 HSV 2 73 o C HSV 1 HSV 2 67 o C

20. 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)

21. Threshold Cycle Microbial Load Testing

22. Concentration log 10 Threshold Cycle Test Sample Threshold Threshold Cycle Threshold Cycle = 35 Load = 10 3.8 copies/ml

23. PRACTICAL 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

24. Sampling Time (Wks) 40 30 20 10 0 Antigenemia Positive cells per 200,000 cells Antigenemia Positive cells per 200,000 cells genome copies q-PCR 1 2 3 4 5 6 7 8 9 10 11 Ganciclovir BMT PATIENT 1 1 2 3 4 5 6 7 8 9 10 11 ROCHE PCR “in house” PCR Antigenemia

25. q-PCR 1 2 3 4 5 6 7 8 9 10 11 12 13 80 60 40 20 0 Ganciclovir Foscarnet BMT PATIENT 2 Sampling Time (Wks) Antigenemia Positive cells per 200,000 cells Antigenemia Positive cells per 200,000 cells genome copies 1 2 3 4 5 6 7 8 9 10 11 12 13 ROCHE PCR “in house” PCR

26. 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 R&D capacity and capital)  High capital equipment costs ($ 50,000 -160,000).

27. 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 - 10 10 copies) Reproducible (CV < 2.0 %) Allows for quantitation of results Software driven operation No more expensive than “in house” PCR ($15/test)

28. 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)

29. Alternative Detection Technology Alternative Detection Technology

30. 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

31. Amplicon molecular beacons A B C FRET real-time real-time Reporter Non-fluorescent Quencher Excitation ANNEALING

32. 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)

33. Multiple 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

34. 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

35. Genechip Array Immobilised capture probes Labeled sample DNA x x x x Conjugated fluorophor Image of array

36. pre (red)/post (green) 1000’s genes/pcr amplified segments robot loaded glass slide microarrays (<200um) share data good controls gene arrays known grid positions hybridise 2 fluor-tag samples illuminated confocal microscope quantitation interpretation

37. Microarrays (Gene Chips) APPLICATIONS Genome mutational analysis Multiple drug resistance Monitor gene expression in cells Pharmocogenomics Screening for multiple infectious agents