Multiplex qPCR

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Introduction & troubleshooting

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Multiplex qPCR

  1. 1. Multiplex qPCR: Assay evaluation & minor troubleshooting By: R.A. Hamidjaja RIVM/Cib/LZO/RBC
  2. 2. Why multiplex qPCR? <ul><li>Capability to do a quantitative assay </li></ul><ul><li>Time needed from ‘question – answer’ is short (no post PCR work) </li></ul><ul><li>Integration of internal control </li></ul><ul><li>Multiple reactions/assays in a single tube </li></ul><ul><li>Less toxic: no need for intercalating agent (carcinogenic) </li></ul>
  3. 3. Work flow <ul><li>Sample (clinical, environmental, food etc.) </li></ul><ul><li>DNA or RNA extraction </li></ul><ul><li>‘ Specific’ enzymatic amplification (PCR) </li></ul><ul><li>Detection (Post PCR) </li></ul><ul><li>Results </li></ul>
  4. 4. PCR (Polymerase chain reaction) dATP dTTP dCTP dGTP DNA Taq DNA polymerase Deoxyribonucleotide triphosphate Primers (Forward & reverse) Temperature: 95 o C = denaturing 55 o C = annealing 72 o C = elongation + 1 cycle ATCGGCTA TAGCCGAT 5’ 5’ 3’ 3’
  5. 5. PCR contd. 95 o C 72 o C 55 o C T: ATCGGCTA 5’ 3’ TAGCCGAT 5’ 3’ TAGCCGAT 5’ 3’ TAGCCGAT 5’ 3’ ATCGGCTA 5’ 3’ ATCGGCTA 5’ 3’ dATP TAGCCGAT 5’ 3’
  6. 6. qPCR (quantitative PCR) dATP dTTP dCTP dGTP DNA Taq DNA polymerase Deoxyribonucleotide triphosphate Primers (Forward & reverse) + Fluorescentlabeled probe ATCGGCTA TAGCCGAT 5’ 5’ 3’ 3’
  7. 7. qPCR contd. 95 o C 72 o C 55 o C T: ATCGGCTA 5’ 3’ TAGCCGAT 5’ 3’ dATP TAGCCGAT 5’ 3’ TAGCCGAT 5’ 3’ TAGCCGAT 5’ 3’ Detector
  8. 8. Multiplex qPCR dATP dTTP dCTP dGTP DNA Taq DNA polymerase Deoxyribonucleotide triphosphate Primers (Forward & reverse) + Fluorescent labeled probes ATCGGCTA TAGCCGAT 5’ 5’ 3’ 3’
  9. 9. Fluorescent label / Dye (1) Absorption of blue light Emission of red light [A] [E] 610 nm detector
  10. 10. Fluorescent label / Dye (2) [E] 610 nm 570 nm 530 nm
  11. 11. Fluorescent label / Dye (2) [E] 610 nm 570 nm 530 nm Spectral overlap
  12. 12. Spectral overlap <ul><li>Spectral overlap is compensated by an algorithm found within the thermocycler software: </li></ul><ul><li>Color compensation </li></ul><ul><li>Pure dye calibration </li></ul>Before compensation After compensation
  13. 13. Data output <ul><li>Amplification curve </li></ul><ul><li>Ct = Cp value (cycle threshold) </li></ul><ul><li>Standaard curve (only when doing quantitative assay) </li></ul>
  14. 14. Amplification curve
  15. 15. Cp calculations <ul><li>1 cycle = 1 temp process of ‘denature-annealing-extension’ </li></ul><ul><li>During qPCR enough fluorescent light need to be present before it can be detected </li></ul><ul><li>The first time fluorescent light can be detected = Cp = Ct value </li></ul><ul><li>Many starting materials = faster accumulation of fluorescent light = lower Cp </li></ul><ul><li>Few starting materials = slower accumulation of fluorescent light = high Cp </li></ul>
  16. 16. Amplification curve Cp=16 Cp=23
  17. 17. Problem discovery (1) <ul><li>Essential tools: </li></ul><ul><li>Quantifiable positive control (e.g.: culture, plasmid modified to contain target sequences, artificial DNA/RNA with complimentary sequences, etc) </li></ul><ul><li>Correct combination of dyes and apparatus (note: excitation and detection) </li></ul><ul><li>Panel of important negative and positive controls (e.g.: host DNA, vector DNA and DNA of similar organisms) </li></ul><ul><li>Properly designed primers and probes suited for your experimental need </li></ul>
  18. 18. Problem discovery (2) <ul><li>Where to look? </li></ul><ul><li>False positive or false negative results (control panel) </li></ul><ul><li>Amplification curve (form en fluorescence intensity) </li></ul><ul><li>Standard curve = amplification efficiency </li></ul><ul><li>Theory = 2 = 100% </li></ul><ul><li>Reality = 1.9 – 2.05 = 95% - 103% </li></ul><ul><li>“ Old school” gel electrophoresis </li></ul>
  19. 19. Amplification curve
  20. 20. Amplification curve
  21. 21. Case #1 Exp: Apr 2008 Exp: Sep 2008
  22. 22. Troubleshoot #1 Problem: New batch of probe is not good Apr 2008 Sep 2008 Okt 2008
  23. 23. Case #2 Channel 670 nm Before CC After CC
  24. 24. Troubleshoot #2 Non optimal dyes/label combination caused non optimal color compensation. Other possibilities: Non optimal probes concentrations Texas Red overlap CFR 590 overlap
  25. 25. Single vs Multiplex (incl. CC)
  26. 26. Detection limit Extraction kit limit: 25 mg tissue (20 pathogens) Extraction: 1 g tissue (100 pathogens) PCR detection limit : 1 pathogen in 5 ul of eluted DNA 100 ul eluted DNA: 20 pathogens 1 g meat sample: 800 pathogens PCR detects: 5 pathogens in 5 ul of eluted DNA 100 ul eluted DNA: 100 pathogens 1 g meat sample: Limit 100 pathogens

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