lý thuyết Realtime

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  1. 1. Trao i trc tuyn ti: http://www.mientayvn.com/Y_online.html
  2. 2. TS. BS. Th Thanh Thy Realtime PCR Nguyn tc v ng dng
  3. 3. PCR
  4. 4. PCR V GII HN CA PCR PCR v pht hin sn phm bng in di trn gel agarose 1. Pht hin im cui (End-Point Detection) 2. c hiu km 3. nhy thp 4. phn gii km 4. Ch phn bit da trn kch thc sn phm PCR 5. Phi x l sau PCR, d ngoi nhim 6. Chy gel nh Ethidium bromide (cht gy ung th)
  5. 5. REALTIME PCR Real-time PCR l phn ng PCR m qu trnh nhn bn DNA c theo di trc tip trn my lun nhit theo tng chu k nhit. Kim sot tn hiu hunh quang pht ra trong mi chu k phn ng (t l vi lng sn phm PCR to thnh trong tng chu k nhit - Real time). Pht hin v nh lng nng sn phm sau mi chu k nhit da trn cng pht hunh quang.
  6. 6. REALTIME PCR
  7. 7. HOT NG CA MY REALTIME PCR Knh lc kch thchKnh lc pht sng Ngun: laser, LED, n tungsten-halogen Detector: CCD (charge coupled device), PMT (photomultiplier tube)
  8. 8. 1 10 102 103 104 105 106 1 10 102 103 104 105 106 Cycle 15 Cycle 19 Cycle 23 Cycle 26 Cycle 29 Cycle 31 Cycle 33 Cycle 34 Cycle 36 Cycle 39 Cycle 41 Cycle 13 Cycle 11
  9. 9. 1 10 102 103 104 105 106 Cycle 15 Cycle 19 Cycle 23 Cycle 26 Cycle 29 Cycle 31 Cycle 33 Cycle 34 Cycle 36 Cycle 39 Cycle 41 Cycle 13 Cycle 11 1 10 102 103 104 105 106 C.41 C.39 C.36 C.33 C.31 C.29 C.34 Mu cha bit [C] Standard curve
  10. 10. PHNG TRNH TNH S COPY/MU
  11. 11. Threshold: im bt u quan st c tn hiu hunh quang pht ra, ti thi im ny tn hiu do sn phm PCR > tn hiu nn. - Ct (Threshold cycle): l s chu k m ti tn hiu hunh quang ca sn phm khuch i vt qua ngng. -ng hc RealtimePCR Phn tch tin trnh phn ng trong mi chu k nhit nh computer THI IM PHT HIN TN HIU
  12. 12. REALTIME PCR
  13. 13. REALTIME PCR Rael time PCR nh lng s copies trong giai on tng ly tha Log view
  14. 14. Primers Taq polymerase dNTP PCR buffer MgCl2 Real-time PCR MIX Target DNA (Template) Real-time PCR mix Cht pht hunh quang 1. Cht nhum khi chn vo si i DNA s pht hunh quang: Ethidium bromide SYBR green 1 2. Probe c hiu c gn cht pht hunh quang Taqman probe. Molecular Beacon Hybridization probe TN HIU HUNH QUANG TRONG REAL TIME PCR
  15. 15. SYBR GREEN I khng SYBR GREEN I Khng pht hunh quang SYBR GREEN I Hunh quang
  16. 16. SYBR GREEN I DNA can tm DNA khong can tm Khong phan biet c san pham ac hieu hay khong ac hieu
  17. 17. Tnh c hiu khng cao. Tm sot sn phm trc khi s dng probe c hiu Thc hin khi PCR c ti u ha, khng c primer dimer, khng c amplicon khng c hiu SYBR GREEN I
  18. 18. PHN BIT SN PHM C HIU (Melting Curve) Mc ch: phn tch tnh c hiu ca sn phm sau khuch i Melting temperature (Tm) ca dsDNA: To ti pt DNA dng si i, si n Ph thuc chiu di v thnh phn nucleotide Thc hin: tng To, DNA tch nhau ra, a To v 55oC. Tng dn nhit , chp hnh tn hiu hunh quang mi 0,5oC.
  19. 19. Melting Curve
  20. 20. PHN BIT SN PHM C HIU (Melting Curve)
  21. 21. REAL TIME PCR S DNG TAQMAN PROBE 1. U IM Probe c hiu Multiplex PCR 2. NHC IM Gi thnh cao
  22. 22. NGUYN TC HOT NG Taq polymerase c hot tnh 5-3 exonuclease
  23. 23. 5 5 3 3 dNTPs DNA Polymerase ben nhiet Primers Tube phn ng R Q Probe 1. Bin tnh 94oC 2. Bt cp v ko di 60oC Taq l R Q NGUYN TC HOT NG
  24. 24. 5 3 RQ 5 3 Q Taq R 52. Phn ct 3. Hon tt tng hp 5 3 Taq R 5 4. Pht hin 5 3 Taq R 5 5 3 1. Ko di mch Taq R RQ Primers c nhit bt cp 60oC v Taqman probe 70oC
  25. 25. F DNA probe CG C A A A G T A T C A T C C C T C C A GG G C G G C C A A T T Q DNA ch F GC A A A G T A T C A T C C C T C C AG Q CG T T T C A T A G T A GGGA GG T A Stem Loop Reporter Dye REALTIME PCR S DNG BEACON PROBE
  26. 26. Giai on bin tnh NGUYN TC HOT NG
  27. 27. Giai on bt cp NGUYN TC HOT NG
  28. 28. Giai on ko di NGUYN TC HOT NG
  29. 29. 1) Reaction efficiency (PCR efficiency): E%, s dng ng cong chun (standard curve). Ti u E 100% th E=2 2) Reproducibility/linearity (R=Corelation coefficiency) s dng gi tr R hay R2 =1 nh gi chnh xc trong thao tc 3) Specificity s dng Melting Curve 4) Contamination s dng NC C KT QU REALTIME PCR
  30. 30. E%=PCR efficiency (90%-105%) R=Corelation coefficiency (>0.990) C KT QU REALTIME PCR E = 10-1/slope E% = (E-1) x 100% E=10-1/slope = 10 -1/-3.425 = 1.959 E% =(1.959 - 1) x 100% = 95,9
  31. 31. E%=PCR efficiency (90%-105%) R=Corelation coefficiency (>0.990) C KT QU REALTIME PCR Y= ax+b Y = a.(log10Sq) + b Y: Ct X: log10Sq Sq: Starting quantity Sq= 10 [(Ct-b)/a]
  32. 32. NG DNG CA REALTIME PCR TRONG CHN ON V NGHIN CU
  33. 33. n gin, nhanh chng, d phn tch kt qu. Trnh c ngoi nhim v khng cn phn tch sau PCR. C th nh lng trong phm vi ln (log 7 n log 8) nhy cao hn so vi PCR. c hiu cao hn so vi PCR Realtime PCR l cng c pht hin tc nhn gy bnh tt nht trong mu bnh phm (vi sinh, k sinh lm sng, vi sinh thc phm) nhy tt Khng ngoi nhim Thch hp cho lm sng c hiu tt Cng c tt nht
  34. 34. Xc nh s lng virus (HIV, HBV, HCV...) Theo di iu tr Xc nh s lng khi u ca cc tc nhn gy bnh khn cp trong mu bnh (St xut huyt, H5N1, H1N1..) Tin on mc nghim trng v tin on kt qu Realtime PCR l cng c nh lng tc nhn gy bnh tt nht trong mu bnh phm Tnh trng nhim khun thc phm: Samonella, Campyllobacter.
  35. 35. Realtime PCR ng dng trong ung th hc lm sng Pht hin s tn lu c tnh trong bnh ung th -Trong bnh bch cu mn dng ty (CML) v bnh bch cu cp dng Lympho (ALL), dng realtime PCR nh lng 2 t hp gen khc nhau gy bnh l p190 v p210. So snh kt qu nh lng trc v sau iu tr gip nh gi mc tn lu c tnh ca bnh.
  36. 36. Realtime PCR trong nghin cu biu hin gen ng dng rt rng ri trong vic nh gi mc biu hin gen. VD: Sau khi t bo c x l vi khng nguyn hay ha cht, tm hiu s p ng ca TB trong mc biu hin ca gen ch.
  37. 37. Cc ng dng khc ca Real-Time PCR Pht hin GMO (genetically modified organism) hay GEO (genetically engineered organism): sinh vt bin i gene. Pht hin SNP (single nucleotide polymorphism) (genotypic/allelic discrimination)