DNA. Real-time PCR PCR. Exicycler 96 RotorGene Q/6000 HPV16 HPV18 IU

Transcription

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2 Real-Time PCR

3 DNA Real-time PCR Exicycler 96 RotorGene Q/6000 PCR HPV16 HPV18

4 Mix1 Mix2 /μl /μl /μl /μl /μl C 1 : International Unit 2 Lot# 3 Freeze & thawing

5 1 MSDS: Material Safety Data Sheets

6 (TaqMan Real-time PCR HPV16 ' FAM HPV16 BHQ1 TexasRed PCR ' ' ' JOE HPV18 BHQ2 BHQ1 HPV18 ' ' in vitro in vitro PCR 1 Validation 2 Conserved 3 Beta2 Microglobulin

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

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

9 HPV18 HPV16 HPV18 HPV16 HPV DNA HPV16 HPV18 1 High-risk HPVs 2 Precancerous 3 Screening 4 Cytology 5 High-grade cervical disease 6 Grade 7 Squamous intraepithelial lesions

10 /l HPV18 HPV16 HPV18 pvalue HPV16 /l HPV18 /l HPV16 1 Analytical Sensitivity

11 Varicella Zoster Virus 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 Herpes Simplex Virus Type1 Herpes Simplex Virus Type2 (DNA) 1 Analytical Specificity 2 Cross Reactivity

12 HPV16 /µl HPV18 HPV16. HPV18 /µl /µl 1 Linear range 2 Precision 3 Variance 4 Intra assay variation 5 Inter assay variation

13 /µl /µl PCR. C t /µl Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV18 1 SD: Standard Deviation 2 CV: Coefficient of Variation 3 C t : Cycle of threshold

14 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV18 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV16

15 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV18 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV18

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

17 C C C C 1 CytoBrush 2 Mucolytic 3 Urethral epithelial swab

18 dntp PCR Mix1 Mix2 HPV18 HPV16 PCR PCR R 2 R^2 e PCR 1 Efficiency (e PCR )

19 DNA (Cat#) Qiagen DynaBio Blood/ Tissue Genomic DNA Extraction Mini Kit QIAmp DNA Blood Mini Kit 1 Cross Contamination

20 1 Sampling 2 Under or Over estimation 3 Duplicate Real-time PCR

21 PCR x x Real-time PCR 1 Run

22 PCR Exicycler96 (Scan) HPV16 FAM HPV18 JOE TexasRed RotorGene 6000 RotorGene Q Orange Green Yellow (Scan) HPV16 FAM HPV18 JOE TexasRed

23 ExiCycler96 Design Experiment Exicycler Run File a. Master HPV16&18 b. Protocol Takapouzist c. Plate /.,

24 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

25 Append Plate Add Probe.1 JOE BHQ FAM BHQ.2 OK TexasRed BHQ

26 E5 B5 B5 E5 Probe Name Assign STD FAM JOE Type Sample TexasRed Sample Name Assign Sample Name

27 OK Plate Protocol Save Exicycler3 Run Master save as

28 .8 Run Exicycler Master Design Experiment (open) Door Run OK

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

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

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

33 Target Sample Range Ok Green Add Orange Target Sample Ok Yellow Green Range

34 Perform Optimisation Before 1 st Acquisition Close

35 Next Start Run

36 OK RotorGene 6000 /µl RotorGene Q

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

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

39 RotorGeneQ RotorGene6000 Analysis Analysis Show

40 Dynamic Tube Slope Correct Auto Scale Linear Scale Threshold Maximize

41 Quantitation Analysis Ct Standard Curve R^2 Efficiency Quant. Results HPV18 JOE Orange

42 HPV16 TexasRed FAM HPV16 HPV18 TexasRed JOE HPV18 JOE FAM HPV18 HPV16 TexasRed HPV18 HPV16 JOE FAM 1 Flowchart 2 Trouble Shooting

43 TexasRed C t > JOE FAM PCR HPV18 HPV16 TexasRed JOE FAM

44 HPV16 Texas R^2> Ct FAM JOE FAM JOE FAM PCR HPV16 Texas

45 HPV18 Texas R^2> Ct JOE JOE FAM JOE FAM PCR HPV18 Texas

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

47 () /ml /l HPV16 WHO WHO HPV18 1 WHO: World Health Organization 2 Viral Loads 3 1 st WHO international Standard for Human Papillomavirus (HPV) Type16 DNA, code: 06/202 & 1 st WHO international Standard for Human Papillomavirus (HPV) Type18 DNA, code: 06/206 4 Genome equivalent

48 Symptom JOE FAM JOE HPV18 FAM HPV16 PCR TexasRed PCR PCR PCR JOE FAM PCR PCR TexasRed JOE FAM DNA

49 Symptom PCR PCR FAM JOE FAM TexasRed JOE PCR PCR Real-time PCR

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