Analisis Rt PCR assay dan aplikasinya untuk syndromic testing

analisis rt pcr

1. Swara Yudhasasmita Product manager diagnostic PT Sciencewerke Analisis Real-time PCR assay dan Aplikasinya untuk Syndromic testing

2. Basic real-time PCR Analisis & Intepretasi data real-time PCR Syndromic testing analisis dengan seegene viewer Hands on analisis Table of content

3. DNA based technique Conventional Advanced

4. Overview Real-time pcr Conventional Advanced more sensitive up to 1 copy/reaction

5. Real-time PCR Real-time pcr use fluorescent day in reaction SYBR Green (single plex) TaqMan probe (Multiplex) Can be monitored in real-time The result: Ct Value melting curve Qualitative or quantitative data

6. SYBR Green Taqman Probe TOCE & MuDT single plex (1 tube = 1 target) Multiplex (1 tube= up to 4 target) 1 channel = 1 target High Multiplex (1 tube= up to 15 target) 1 channel = 3 target Syndromic testing

7. Basic real-time PCR Analisis & Intepretasi data real-time PCR Syndromic testing analisis dengan seegene viewer Hands on analisis Table of content

8. Melting curve is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature Can be done by Intercalacting dye (SYBR GREEN) 1 melting curve = 1 amplicon Melting curve analysis Data Analysis real-time PCR

9. Melting Temperatures varies from DNA’s of different species based on the GC content, more GC content more the temperature required to melt. The reason for this is due to the presence of 3 hydrogen bonds in the GC Pairing as compared to two hydrogen bonds in the AT pairing. So more energy is to break the GC pair SNP Analysis Primer specificity Two peaks will occur when the primer bind unspecificly. Data Analysis real-time PCR

10. Data Analysis real-time PCR Ct Value : the number of cycles needed to replicate enough DNA/RNA to be detected (crosses a threshold line) Lower ct value > high titer DNA High Ct value > lower titer DNA

11. Real-time PCR application Qualitative detection Quantitative detection Pathogen detected? Yes No Pathogen detected? How many? Yes No IU/mL Copies/reaction

12. Quantitative detection

13. Data Analysis real-time PCR The threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels. threshold can be adjusted : manual (make sure threshold is above background) automatic by software Bad treshold Good treshold

14. Critical points in real-time PCR assay Avoid contamination by putting the NTC & NEC Check the bubble are not present in the tube before run Avoid freeze thawing cycle Do one way workflow

15. Basic real-time PCR Analisis & Intepretasi data real-time PCR Syndromic testing analisis dengan seegene viewer Hands on analisis Table of content

16. Introducing “Syndromic Molecular Diagnostics” in healthcare institutions can enhance the quality of medical care and cost-effectiveness, ultimately leading to improved patient satisfaction. 1. Enhanced Medical Quality : Syndromic molecular diagnostics assist in more accurate disease diagnosis, enabling early detection and initiation of treatment, thus improving patient outcomes. 2. Improved Patient Satisfaction : Accurate and timely diagnosis contributes to higher patient satisfaction by avoiding unnecessary procedures and facilitating faster recovery. 3. Guidance for Treatment : Syndromic molecular diagnostics provide insights into specific disease types and treatment options, aiding in the development and application of personalized treatment approaches. 4. Cost-effectiveness : Accurate diagnosis reduces unnecessary tests and treatments, leading to cost savings for both patients and healthcare institutions. What is syndromic testing?

17. Fever Cough Fatigue Shortness of breath Viral infection? Bacterial infection? Co- infection? Syndromic Testing is necessary to identify exactly the pathogen that cause the disease, Able to help clinician to give the right treatment help patient to get recovery more quickly

18. Timely patient management, improving the patient's quality of life and reducing overall medical costs Syndromic test can prevent serious side effects including infection control failure due to increased resistance due to drug misuse. Benefits of Syndromic MDx Approach from patient management perspective

19. Particularly in case of persistent chlamydial infection where anti-apoptotic mechanisms are activated, thus initiating carcinogenesis that is magnified alongside HPV Co-infection of HPV with STI can lead to syndemic effects on cervical lesions Syndromic MDx Assays: STI, Vaginitis and HPV Wong et al., HPV Titre , Co-Infections and Cervix Cytopathology / doi : 10.14744/ejmo.2022.38937

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21. WORKSHOP Analisis real-time PCR & aplikasinya untuk syndromic testing

22. IVD Kits RuO Kits All process is already validated by the manufacture sample collection device sample preparation & storage extraction kit tube PCR (white/clear) real-time PCR machine Provide analytical performance LoD clinical validation cut off & Threshold RuO : research use only It does not provide data validation for all process. Laboratory should do an internal validation process

23. Bentuk 7 kelompok Masing masing kelompok melakukan analisa : berapa treshold yang diadjust berapa nilai ct value yang muncul Kelompok 1 Kelompok 2 Kelompok 3 Kelompok 4 Kelompok 5 Kelompok 6 Kelompok 7

24. perhatikan hasil nilai ct pada tabel Ct 5.79, 3.44, 3.33 perlu dilakukan adjustment treshold. Pilih salah satu channel adjust treshold hingga melewati noise baseline klik kanan pada kurva lalu pilih show the treshold value nilai Ct pada FAM hilang karena treshold diatur. lakukan hal serupa pada channel lainnya.

25. Kelompok FAM HEX Cal Red 610 Quasar 670 Quasar 705 1 2 3 4 5 6 7

26. 1 Pilih setting>Baseline setting>No baseline substraction

27. 2 Pilih step number 3 lalu Pilih export > Export All data sheets > Excel 2007 buat folder di dekstop HPV analisis Quanstep3 Quanstep4 Quanstep5 simpan file export sesuai dengan step number yang dipilih

28. 3 Ulangi langkah sebelumnya untuk step number 4 dan 5

29. Analisis seegene viewer Buka Seegene viewer Pilih assay allplex HPV 28

30. Analisis seegene viewer Pilih open, lalu pilih folder quantstep 3 Pilih file excel quantification cq result

31. set A set B set A set B

32. Analisis seegene viewer row 2 pilih untuk set A Row 6 pilih untuk set B row 11 A-C : set A row 11 F-H : Set B

33. QC Checking check PC dan NC sesuai kriteria pada tabel Check IC pada masing masing sample.

34. Pilih sample sesuai dengan kelompok. Positif HPV subtype apa? Nilai Ct?

35. Swara Yudhasasmita Product manager diagnostic Thank You “Making science work for you”