Sommaire dosage par PCR en temps réel

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1 Sommaire dosage par PCR en temps réel
Rappel biochimique

2 Evolution of Molecular Detection Technologies
Non-Amplified Methods (DNA-Hybridization-Assays) Conventional PCR Assays (EIA / Chemiluminescence detection) Real-Time PCR Assays poor sensitivity long processing time labor-intensive improved sensitivity limited dynamic range limited automation high sensitivity broad dynamic range easily automated

3 Definition de la Polymerase Chain Reaction
Définition de la PCR Definition de la Polymerase Chain Reaction Méthode in vitro simple, rapide, sensible, et universelle qui permet d’amplifier des séquences/régions d’ADN/ARN à partir d’une source complexe d’acides nucléiques de maniére sélective.

4 2 oligonucléotides comme amorces
Principe de la PCR 50 60 70 80 90 100 ADN double brin ADN-Polymerase 2 oligonucléotides comme amorces dNTP’s tampon (co-facteur) Température ambiante

5 Principe de la PCR Dénaturation (1) 50 60 70 80 90 100 94ºC

6 Principe de la PCR Extension amorce (3) Primer Annealing (2) 72ºC 60ºC
50 60 70 80 90 100 72ºC 50 60 70 80 90 100 60ºC 50 60 70 80 90 100 72ºC Pol Pol

7 Principe de la PCR AND doublé 50 60 70 80 90 100 72ºC

8 Amplification exponentielle
Principe de la PCR Amplification exponentielle 4ème cycle Gene recherché 3ème cycle 2nd cycle 1er cycle 35tème cycle ADN matrice 21 = 2 copies 22 = 4 copies 235 = 34 milliard copies 8 copies 16 copies

9 Détection des produits de PCR
3 modes de detection : Gels Agarose (point-final, Bromure d’éthidium) Immunologiquel (point-final, amorce & sonde marquées comme antigène) Real-time (temps réel, sonde fluorescentes)

10 Pas de différenciation possible entre ces signaux !
Detection Agarose Gel Pas de différenciation possible entre ces signaux ! PCR classique: Détection au point final sur gel d‘Agarose- 5 10 15 20 30 40 50 Plateau Phase Linéaire Intensité Fluorescence Phase Exponentelle Nombre de Cycles

11 Detection Méthode Immunologique
PCR classique: Analyse au point final e.g. LCx, Cobas Amplicor Anti-A AP Micro- particules Mu MUP + P A C Plateau Phase Linéaire Intensité Fluorescence Exponential Phase Nombre de Cycles

12 Detection Méthode Temps réel
Plateau Phase linéaire Intensité Fluorescence Phase exponentielle Seuil Nombre de Cycles

13 Detection Méthode Temps réel
Détermine le QUAND du signal plutôt que l‘intensité du signal (analyse au point final) La mesure du QUAND est obtenu PCR en temps réel par détermination du Ct Ct = cycle seuil: Le nombre de cycle calculé auquel le produit PCR croise un seuil de détection Ct Seuil

14 Real-Time PCR Detection
Real-Time PCR Mesure en phase Exponentielle Mesure de 96 Repliques identiques Real-Time PCR Detection Detection PCRclassique (detection par EtBr-Gel / EIA / Chemiluminescence) phase Plateau faible pracision faible sensibilité plage dynamique pas étendue (~2 logs) phase exponentielle grande précision Ligne de base Phase linéaire Nombre de cycle

15 Principe des tests RealTime
Utilisation d’une sonde d’hybridation Séparation de l’amplification et de la lecture: Ajouter une étape supplémentaire à 35°C dans chaque cycle de PCR Pour l’hybridation de la sonde et la lecture Again, the most important difference for our assays is the uncoupling of the propbe hybridization&reading step from the extension step. By having the option to chose a lower hybridization temperature of 35 °C, our probes bind much more efficiently to target sequences with mismatches. This greatly enhances our mismatch tolerability of our RealTime HIV-1 and HCV assays. TaqMan assays do not have this capability. Here, the probe needs to be cleaved by the polymerase and therefore needs to bind to the target during the extension step which happens at around 55 °C.

16 Principe des tests RealTime
DENATURATION Again, the most important difference for our assays is the uncoupling of the propbe hybridization&reading step from the extension step. By having the option to chose a lower hybridization temperature of 35 °C, our probes bind much more efficiently to target sequences with mismatches. This greatly enhances our mismatch tolerability of our RealTime HIV-1 and HCV assays. TaqMan assays do not have this capability. Here, the probe needs to be cleaved by the polymerase and therefore needs to bind to the target during the extension step which happens at around 55 °C.

17 Principe des tests RealTime
Hybridation des primers Et élongation Again, the most important difference for our assays is the uncoupling of the propbe hybridization&reading step from the extension step. By having the option to chose a lower hybridization temperature of 35 °C, our probes bind much more efficiently to target sequences with mismatches. This greatly enhances our mismatch tolerability of our RealTime HIV-1 and HCV assays. TaqMan assays do not have this capability. Here, the probe needs to be cleaved by the polymerase and therefore needs to bind to the target during the extension step which happens at around 55 °C.

18 Principe des tests RealTime
1 Q R Q R 2 R Q Hybridation de la sonde ET LECTURE Again, the most important difference for our assays is the uncoupling of the propbe hybridization&reading step from the extension step. By having the option to chose a lower hybridization temperature of 35 °C, our probes bind much more efficiently to target sequences with mismatches. This greatly enhances our mismatch tolerability of our RealTime HIV-1 and HCV assays. TaqMan assays do not have this capability. Here, the probe needs to be cleaved by the polymerase and therefore needs to bind to the target during the extension step which happens at around 55 °C. R 3

19 Emplacement du pack réactif sur le m2000sp
Configuration du kit réactif 4amorces, 2 Sondes, dNTP‘s Emplacement du pack réactif sur le m2000sp Enzyme Activateur This slide illustrates the RealTime HIV and HCV kit configuration for the reagents. 1 Reagent Kit contains 4 packs for 24 reactions per pack which equals 96 testpoints in total. One pack, 24 reagents, consits of three different bottles, on bottle containing the polymerase enzyme (rTth), on bottle containing primers and nucleotides, and one bottle containing the activator (Manganese) for the polymerase enzyme. Staring a PCR reaction is as easy as pipetting all three bottles into one common mastermix-tube, and the adding the extracted sample and a certain amount of mastermix to the 96-well plate. Having done this, you only would need to put the plate into the m2000rt instrument and press ‚Go‘. 24 tests (1 Kit: 4 x 24 tests) Séries de 24, 48, (72, 96)

20 m2000 HIV-1 Assay Description: Oligo Mix Reagent
PRIMERS The assay utilizes two distinct sets of primers; a set specific for HIV and another set specific for the internal control Non-competitive for primers, can detect IC even at high levels of HIV The HIV-1 Primers target the integrase gene in the pol region The IC Primer sequences are unrelated to the analyte sequences (target is a plant gene) PROBES An HIV specific probe and an internal control specific probe, each labeled with a different fluorophore at the 5’-end, are used to detect amplified product at each cycle HIV probe is a long partially double stranded probe (FAM labeled) IC probe is a short single stranded probe (VIC labeled)

21 Partially Double Stranded Probe Design for HIV Probe
Q In the absence of target, the probe hybridizes to the quencher probe. The energy from the excited fluorophore is absorbed by the quencher on the quencher probe, preventing fluorescent signal generation Emission Excitation In the presence of target, the probe prefers to hybridize to the target sequence, dissociates from the quencher probe, allowing fluorescence detection. R

22 Short Single Stranded Probe Design for IC Probe
Q In the absence of target, the energy from the excited fluorophore is absorbed by the quencher at the 3’-end of the probe, preventing fluorescent signal generation. Emission Excitation In the presence of target, the probe hybridizes to its complementary sequence, separating the two ends of the molecule, allowing fluorescence detection. Q R

23 m2000 HIV-1 and HCV Assay Description: Calibrator/Control Source
Armored RNA is used as the Calibrator and Control Material Armored RNA has long-term stability in plasma RNA is encapsulated in bacteriophage protein that protects the RNA from nuclease digestion. The RNA is released during the sample prep procedure

24 m2000 HIV-1 and HCV Assay Description: Calibrators
Assay calibrators are Armored RNA in negative plasma. Assay calibrators (2 levels in replicates of 3) are processed through the same sample preparation procedure as specimens Calibration curve is stored on the instrument; recalibration is required when reagent lots are changed Sample quantitation is achieved through linear extrapolation of a 2 level calibration curve to the upper limit of quantitation (ULQ) and to the assay threshold Assay response is linear throughout the dynamic range

25 m2000 HIV Assay Description: Calibration Curve and Sample Quantitation
y = x 5 10 15 20 25 30 35 1 2 3 4 6 7 8 Observed log copies/ml Cycle Calibrators aRNA Dilutions Cal A Cal B

26 m2000 HIV-1 and HCV Assay Description: Controls
One negative control and two positive controls are run on each run, and are processed through same sample preparation procedure as specimens Internal Control is added to each sample, including controls and calibrators, at the start of sample preparation through the lysis buffer Internal Control is used to evaluate sample validity and functions as a check for recovery and inhibition, and does not serve as a Quantitation Standard

27 m2000 HIV-1 Assay Description: Results Reported
All Valid results reported as Not Detected (below assay thresholds) Detected, < 40 copies/ml Concentrations from 40 to 10 million copies/ml > 10 million copies/ml m2000 HIV-1 RNA Quantitative assay is standardized to VQA and results reported as copies/ml or log copies/ml.

28 PERFORMANCE Sensibilité 40 copies/ml Plage linéaire
40 à 10 millions copies/ml Spécificité >99% Détection des sous types Tous les sous types du groupe M, groupe N et groupe O Échantillons Plasma ACD et EDTA Volume 1.0 ml (0.2 ml et 0.5 ml); 0,6ml Contrôle interne ARN encapsulé Calibration 2 points Précision <0.5 log copies/ml ( I.C. À 95 %) (E.T. <0,15) Préparation échantillon m1000 Durée ontention des résultats 5.5 heures Format de rendu de résultats copies/ml, log copies/ml, IU/ml, log IU/ml Standardisation Standard VQA Région ciblé Région Intégrase conservée du gène pol * Marqué CE en Protocole 0,5 ml sensibilité 75 copies/ml Protocole 0,2 ml sensibilité 150 copies/ml

29 OUVERTURE