Introduction & troubleshooting

1. Multiplex qPCR: Assay evaluation & minor troubleshooting By: R.A. Hamidjaja RIVM/Cib/LZO/RBC

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. 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. 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. 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. 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. Fluorescent label / Dye (1) Absorption of blue light Emission of red light [A] [E] 610 nm detector

10. Fluorescent label / Dye (2) [E] 610 nm 570 nm 530 nm

11. Fluorescent label / Dye (2) [E] 610 nm 570 nm 530 nm Spectral overlap

14. Amplification curve

16. Amplification curve Cp=16 Cp=23

19. Amplification curve

20. Amplification curve

21. Case #1 Exp: Apr 2008 Exp: Sep 2008

22. Troubleshoot #1 Problem: New batch of probe is not good Apr 2008 Sep 2008 Okt 2008

23. Case #2 Channel 670 nm Before CC After CC

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. Single vs Multiplex (incl. CC)

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