(DNA) Real-time PCR. Exicycler 96 Rotor-Gene Q/6000 PCR

Transcription

1

2 Real-Time

3 (DNA) Real-time Exicycler 96 Rotor-Gene Q/6000 IU

4 Mix1 Mix2 IU/μl IU/μl IU/μl IU/μl IU/μl IPC NTC C 1 Lot# 2 Freeze & thawing

5 1 MSDS: Material Safety Data Sheets

6 (TaqMan Real-time ' FAM ' BHQ1 ' BHQ1 ' JOE in vitro in vitro 1 Validation 2 Conserved

7 Real-Time Real-time Real-time Real-time SYBR Green DNA (DNA) (DNA) 1 Post 2 UV Transilluminator 3 Non Specific Methods 4 Signal

8 TaqMan Real-time MRD 1 Specific Methods 2 Minimal Residual Disease

9 (DNA) HBV HBV HBe (IU) IU/ml IU/l 1 Enveloped DNA Virus 2 Hepadnaviridae 3 End-stage Liver Disease 4 Hepatocellular Carcinoma 5 Copy 6 Occult HBV 7 IU: International Unit 8 WHO: World Health Organization 9 Viral Loads 10 2 nd WHO International Standard for Hepatitis B Virus DNA Nucleic Acid Amplification Techniques, code: 97/750.

10 pvalue IU/l IU/l HBV IU/l 1 Analytical Sensitivity

11 G A WHO A1 A2 B2 B4 C2 D1 D3 E F2 G Log 10 IU IU 1 Analytical Specificity 2 1 st WHO International Reference Panel for Hepatitis B Virus Genotypes for Nucleic Acid Amplification Techniques -Based Assays

12 Human Herpes Simplex virus type 1 Human Herpes Simplex virus type 2 Varicella Zoster Virus (Type B) Human Cytomegalovirus Epstein-Barr Virus 2nd WHO International Standard for Parvovirus B19 DNA for Nucleic Acid Amplification (NAT) Assay WHO 2nd International Standard For Hepatitis C Virus RNA for Genomic Amplification Technology Assays (DNA) IU/µl IU/µl IU/µl HBV. 1 Cross Reactivity 2 Linear range

13 IU/µl IU/µl. C t IU/µl Ct Ct CV Ct IU IU CV IU Intra Assay variation Inter Assay variation Total variance Exicycler96 1 Precision 2 Variance 3 Intra assay variation 4 Inter assay variation 5 SD: Standard Deviation 6 CV: Coefficient of Variation 7 C t : Cycle of threshold

14 Ct Ct CV Ct IU IU CV IU Intra Assay variation Inter Assay variation Total variance Exicycler96 Ct Ct CV Ct IU IU CV IU Intra Assay variation Inter Assay variation Total variance Rotor Gene Q Ct Ct CV Ct IU IU CV IU Intra Assay variation Inter Assay variation Total variance Rotor Gene Q

15 HBV IU/ml. 1 Diagnostic Evaluation 2 Clinical Sensitivity 3 Clinical Specificity

16 Roche Cobas Taqman HBV 1 Diagnostic Correlation

17 Real-time (Heating-Block) DNA rpm Real-time 1 DNase RNase Free 2 Vortex 3 Spin 4 Strip 5 Polymerase Chain Reaction ()

18 EDTA g 1 E.D.T.A 2 Citrated

19 C C C mg/dl mg/dl IU/ml

20 dntp Mix1 Mix2 IU R 2 R^2 e IPC) C t 1 Efficiency (e ) 2 Internal Positive Control

21 Real-time Real-time HBV N.T.C FAM HBV JOE C t 1 No Template Control

22 DNA Roche (Cat#) DNA/RNA DynaBio Viral Nucleic Acid Extraction Mini Kit High Pure Viral Nucleic Acid Kit (DNA) Elution Buffer 1 Cross Contamination 2 Multiplex

23 Real-time 1 Sampling 2 Under or Over estimation 3 Duplicate

24 x x Real-time 1 Run

25 Exicycler96 HBV (Scan) FAM JOE RotorGene 6000 RotorGene Q HBV (Scan) FAM JOE

26 ExiCycler96 Exicycler Run Design Experiment File a. Master HBV b. Protocol Takapouzist c. Plate /.,

27 Append Incubate Temperature: 95 Temperature: 95 Temperature: 58 Time: 5 min Append Time: 15 sec Append Time: 50 Sec Append a. Line=2 b. Cycle=50 Temperature: 72 Append Scan Incubate Time: 20 Sec Append Go to

28 Append JOE BHQ FAM BHQ Plate Add Probe OK.1.2 B5.3

29 E5 B5 E5 Probe Name Assign.4 Type Sample Name Assign Sample Name.5

30 OK Plate Protocol Save Exicycler3 Run Master save as

31 Design Run Exicycler Master Experiment (open).8 Door Run OK

32 RotorGene Q RotorGene 6000 New Locking Ring Attached New Quick Start 36-Well Rotor Next Next

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34 Gain Optimisation Auto-Gain Optimisation Set Up OK Green Auto-Gain Optimisation Set Up Add

35 Add Target Sample Range Ok Yellow Auto-Gain Optimisation Set Up Add

36 Target Sample Range Ok Green Perform Optimisation Before 1 st Acquisition Close

37 Next Start Run

38 RotorGene 6000 IU/µl RotorGene Q

39 File Exicyler96 Exicycler Run Exicycler Analysis Open Open Absolute Quantification Ok

40 Trend Average Sub. Method Threshold Level Flu. Graph FAM JOE STD Curve R^2 Efficiency Ct Report Form

41 RotorGeneQ RotorGene6000 Analysis Analysis Show

42 Dynamic Tube Slope Correct Auto Scale Linear Scale Threshold Maximize

43 Quantitation Analysis Ct Standard Curve R^2 Efficiency Quant. Results JOE

44 HBV (DNA) JOE FAM FAM JOE FAM C t HBV JOE FAM 1 Flowchart 2 Trouble Shooting

45 JOE JOE ± FAM FAM R^2> Ct FAM HBV JOE IU/ml

46 IU IU IU/l (WHO) Result (IU/ml)= Result (IU/µl) Elution Volume (µl) Sample Volume (ml) IU/µl (IU/ml) = IU/ml R^2 e 1 PBS

47 FAM HBV JOE FAM Symptom FAM JOE DNA Ct ± FAM

48 Symptom FAM Real-time

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