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

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The Application of Real-Time PCR in the Diagnosis of Infectious Disease

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  1. The Application of Real-Time PCR in the Diagnosis of Infectious Disease T.P.Sloots Clinical Virology Research Unit, RCH, & Microbiology, QHPS.

  2. Disadvantages of PCR • Technically demanding • Can be expensive • Risk of contamination • Need rigid QC 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 = < 24 hrs)

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

  4. 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

  5. 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

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

  7. 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

  8. 3. 1. 2. 4. real-time real-time LightCycler real-time Set Up

  9. real-time real-time LightCycler real-time Hardware

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

  11. real-time real-time LightCycler real-time Operation

  12. 95oC Denaturation

  13. Excitation FRET P Fluorimeter Reading Emission Tm 55oC Primer/Probe Annealing

  14. 72oC Primer Extension

  15. real-time real-time LightCycler real-time Applications • Detection of Infectious Disease agents • Target Characterisation • Determining Microbial Load (quantitation)

  16. 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

  17. Hybridisation probes (to HSV-1) HSV-1 no mismatch Amplicon HSV-2 mismatch real-time real-time LightCycler real-time Characterisation of HSV by melting curve Application HSV DNA pol Primers common to HSV 1 & 2

  18. Melting Curve Analysis 55oC HSV 2 HSV 1 HSV 1 67oC HSV 2 73oC HSV 1 HSV 2 HSV 1 HSV 2

  19. 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)

  20. Threshold Cycle real-time real-time real-time real-time PCR quantitation Microbial Load Testing

  21. Threshold Cycle = 35 Load = 103.8 copies/ml Test Sample Threshold Threshold Cycle Threshold Cycle Concentration log 10

  22. 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

  23. 1 2 3 4 5 6 7 8 9 10 11 ROCHE PCR “in house” PCR 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)

  24. 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)

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

  26. 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 < 2.0 %) • Allows for quantitation of results • Software driven operation • No more expensive than “in house” PCR ($15/test)

  27. 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)

  28. Alternative Detection Technology

  29. 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

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

  31. 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)

  32. Multiple DNA Arrays • Detection of thousands of gene sequences simultaneously • Capacity for minitiarisation • Suitable for automation • Enormous analytical power

  33. 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

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

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

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

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