CT = control s = sample

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Bethanie Lynch
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1 PANFLAVIVIRUS RT-qPCR Trainer: Dr. Cristina Domingo Assistants: Pranav Patel/Ravish Paliwal 1. Thaw all reagents except the Enzyme Mix (keep at -20 C) 2. Prepare maser mix for the RT-qPCR assay (NO DNA SITE) One step RT-qPCR Reagents: AgPath-ID TM One-Step RT-PCR (AB) Final Volume [µl] 25 Vol. [µl] Factor Mix+Excess 1x 14x Excess 10% H 2 O 1, ,1 2x PCR buffer 12, ,5 Primer/probe mix (10µM) ,6 Detection Enhancer ,4 Enzyme-Mix ,4 Master Mix Volume 20 Template 5 3. Distribute 20 µl of master mix in each tube (NO DNA SITE) 4. Distribute 5 µl sample in the following order (DNA SITE) CT- s1 s2 s3 s4 s5 s6 CT+ CT = control s = sample 5. Cap the tubes 6. Distribute 5 µl of standard samples from 10 1 to Start with the lowest concentration. 7. Cap the tubes 8. Spin down 9. Program the thermocycler and start your PCR Reaction Step Time [sec] Temp [ C] Cycles RT PCR * 45 *Fluorescence measurement 10. *Fluorescence measurement 11. Check the results. Check that the baseline and threshold cycle are ok. Establish the baseline and the threshold cycle regarding your negative control if needed. 12. Analyze the results 13. Print a report for your records ANALYSIS OF RESULTS The controls validate the assay? Yes or No Standard Curve results

2 Individual samples results S1 S2 S3 S4 S5 S6 ADDITIONAL INFORMATION PRIMERS and PROBE RT-qPCR To order in TiB Molbiol please refer to PanFlavi primer/probe set RKI training course Pranav Patel et al, (unpublished results). See manuscript in the Molecular Practices Section of your folder STANDARD CURVE The assays are provided with a standard curve provided in 6 different quantities to yield from 10 1 to 10 6 target molecules in 5 l once dissolved. Start always with the lowest concentrated standard. The standards are provided in a row of tubes with a foil lid. To prepare the standard solution you have to punch a hole through the sealing foil, add 40 l PCR-grade water to each vial, mix by pippeting the solution up and down 10 times. Use only 5 l for a 20 l PCR reaction mix. The rest of the solution you could save at 4 C for short time. When store longer they can not be used as quantification references anymore, but like qualitative controls of your PCR. Take into account that preparation of standards and manipulation of the solutions may cause contamination of the working-place (aerosol)

3 Generic FLAVIVIRUS RT-heminested-PCR Trainer: Dr. Cristina Domingo Assistants: Pranav Patel/Ravish Paliwal 1. Thaw all reagents except the RT-mix (keep at -20 C) 2. Prepare maser mix for the RT-PCR assay (NO DNA SITE) One step RT-PCR Reagents: QIAGEN One Step RT-PCR kit Final Volume [µl] 25 Vol. [µl] Factor Excess [%] RT-PCR-Protocol 1x 8 10 Water 6,40 51,2 56,3 5x RT-PCR mix 5,00 40,0 44,0 dntps mix 1,00 8,0 8,8 cfd2 primer [10 µm] 1,80 14,4 15,8 MAMD Primer [10 µm] 1,80 14,4 15,8 RT-mix 1,00 8,0 8,8 Master Mix Volume 17 RNA 8 3. Distribute 17 µl of master mix in each tube (NO DNA SITE) 4. Distribute 5 µl sample in the corresponding tubes in the following order (DNA SITE) CT- s1 s2 s3 s4 s5 s6 CT+ CT = control s = sample 5. Cap the tubes 6. Spin down 7. Program the thermocycler and start your PCR Reaction Step Time [sec] Temp [ C] Cycles RT PCR Final extension ~ 4 8. Prepare the heminested PCR Master Mix (NO DNA SITE) Nested PCR Final Volume [µl] 50,00 Vol. [µl] Factor Excess [%] PCR-Protocol: 1x 8 10 Water 35,10 280,8 308,9 10x buffer 5,00 40,0 44,0 dntps 25mM 0,40 3,2 3,5 MgCl2 50mM 1,50 12,0 13,2 cfd2 primer [10 µm] 1,25 10,0 11,0 FS778 primer [10 µm] 1,25 10,0 11,0 Taq polymerase 0,50 4,0 4,4 Master Mix volume 45,00 DNA first round 5,00

4 9. Distribute 45 µl of master mix in each tube (NO DNA SITE) 10. Distribute 5µl of the RT-PCR round in the corresponding tubes in the following order (nested PCR SITE) CT- s1 s2 s3 s4 s5 s6 CT+ 11. Cap the tubes 12. Spin down 13. Program the thermocycler and start your PCR Reaction Step Time [sec] Temp [ C] Cycles Denaturing ,00 94 PCR 60, ,00 72 Final Extension 600,00 72 ~ Run 10µl of PCR product plus 5µl loading buffer on a 1.5% Agarose gel (30 min; 100V) during 30 minutes to visualize a 250 pb. band 15. Take a photograph for your records ANALYSIS OF RESULTS The controls validate the assay? Yes or No Individual samples results S1 S2 S3 S4 S5 S6

5 ADDITIONAL INFORMATION PRIMERS RT-PCR Primer MAMD Primer CFD2 5 - AACATGATGGGRAARAGRGATAA GTGTCCCAGCCGGCGGTGTCATCAGC-3 * PRIMER heminested-pcr Primer FS AARGGHAGYMCDGCHATHTGGT-3 REFERENCES 1. Scaramozzino N, Crance JM, Jouan A et al. Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J Clin Microbiol May;39(5):1922-7

EXPECTED RESULTS SAMPLE DENGUE SPECIFIC PANFLAVI GENERIC CONCLUSION CT+/CT- POS/NEG POS/NEG EXPERIMENT VALIDATED S1 NEGATIVE POSITIVE OTHER FLAVIVIRUS/ SEQUENCING REQUIRED S2 POSITIVE POSITIVE

6 EXPECTED RESULTS SAMPLE DENGUE SPECIFIC PANFLAVI GENERIC CONCLUSION CT+/CT- POS/NEG POS/NEG EXPERIMENT VALIDATED S1 NEGATIVE POSITIVE OTHER FLAVIVIRUS/ SEQUENCING REQUIRED S2 POSITIVE POSITIVE DENGUE/ SEROTYPING REQUIRED S3 NEGATIVE NEGATIVE NO FLAVIVIRUS S4 NEGATIVE POSITIVE OTHER FLAVIVIRUS/ SEQUENCING REQUIRED S5 POSITIVE POSITIVE DENGUE SEROTYPING REQUIRED S6 POSITIVE POSITIVE DENGUE/ SEROTYPING REQUIRED

SuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit

SuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit Cat. No. H0521 Size: 2 sets (22 Vβ families/each, with enzymes) H0522 Size: 4 sets