(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 Real- (TaqMan 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 1 Herpesviridae 2 Chicken pox 3 Shingles 4 Self-limiting 5 Immunosuppressed 6 Pneumonia 7 Encephalitis

10 pvalue Copy/l Copy/l VZV Copy/l 1 Analytical Sensitivity

11 Human Herpes Simplex virus type 1 Human Herpes Simplex virus type 2 Human Cytomegalovirus 2nd WHO International Standard for Hepatitis B DNA for Nucleic Acid Amplification (NAT) Assay 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 Human Papilloma Virus Type16 Human Papilloma Virus Type18 (DNA) 1 Analytical Specificity 2 Cross Reactivity

12 Coies/µl Copy/µl Copies/µl VZV. Copies/µl Copies/µl 1 Linear range 2 Precision 3 Variance 4 Intra assay variation 5 Inter assay variation

13 . C t Copy/µl Ct Ct CV Ct Copy Copy CV Copy Intra Assay variation Inter Assay variation Total variance Exicycler96 Ct Ct CV Ct Copy Copy CV Copy Intra Assay variation Inter Assay variation Total variance Exicycler96 1 SD: Standard Deviation 2 CV: Coefficient of Variation 3 C t : Cycle of threshold

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

15 VZV. 1 Diagnostic Evaluation 2 Clinical Sensitivity 3 Clinical Specificity

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

17 EDTA g 1 E.D.T.A

18 C C C mg/dl mg/dl IU/ml

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

20 Real-time Real-time VZV N.T.C FAM VZV JOE C t 1 No Template Control

21 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

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

23 x x Real-time 1 Run

24 Exicycler96 VZV (Scan) FAM JOE RotorGene 6000 RotorGene Q VZV (Scan) FAM JOE

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

26 Append Incubate Temperature: 95 Temperature: 95 Temperature: 56 Time: 5 min Append Time: 15 sec Append Time: 50 Sec Append Temperature: 72 a. Line=2 b. Cycle=50 Append Scan Incubate Time: 20 Sec Append Go to

27 Append JOE BHQ FAM BHQ Plate Add Probe OK.1.2

28 E5 B5 B5 E5 Probe Name Assign Type Sample Name Assign Sample Name

29 OK Plate Protocol Save Exicycler3 Run Master save as

30 .8 Run Exicycler Master Design Experiment (open) Door Run OK

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

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

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

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

36 Next Start Run

37 RotorGene 6000 Copy/µl RotorGene Q

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

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

40 RotorGeneQ RotorGene6000 Analysis Analysis Show

41 Dynamic Tube Slope Correct Auto Scale Linear Scale Threshold Maximize

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

43 VZV (DNA) JOE FAM FAM JOE FAM C t VZV JOE FAM 1 Flowchart 2 Trouble Shooting

44 JOE JOE ± FAM FAM R^2> Ct FAM VZV JOE Copies/ml

45 Copy Copy/l Copy Result (Copies/ml) = Result (Copies/µl) Elution Volume (µl) Sample Volume (ml) Copies/µl (Copies/ml) = Copies/ml R^2 e 1 PBS

46 FAM VZV JOE FAM Symptom FAM JOE ± Ct DNA FAM

47 Symptom FAM Real-time

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