TITLE: IPMC SARS-CoV-2 One-Step qPCR Protocol on BIOMARK
AUTHORS: Julien Fassy, Caroline Lacoux, David Rouquié, Jean Louis Nahon, Pascal Barbry, Laure-Emmanuelle Zaragosi, Bernard Mari

[DESCRIPTION]
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[STEPS]
?. [Sample processing upon arrival]
To be performed in the appropriate biosafety conditions (BSL2 laboratory)Transfer the totality of the transport medium into a 2 mL cryotube and stored at -80°C in a hermetically sealed body bag.If performing RNA extraction prior to RT-PCR, proceed to steps 2 to 8.If performing RT-PCR on samples without RNA extraction, go directly to step 9.
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
Add 560 Buffer AVL. Close the tube caps and vortex for >15 s.  Note: If using a volume of sample other than 140 µL, increase the amount of Buffer AVL proportionally.
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
lncubate for 10 min
0 Room temperature
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
Briefly centrifuge the tube to remove drops from the inside of the lid
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
Tubes can be thouroughly wiped with PHAGO’SPRAY ND (Phagogene) or Virospray (Sterisciences) and taken out the BSL2 laboratory.
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
Go to Qiacube.  Elution volume: default 60 µL of AVE buffer.See Qiacube protocol in the attached file.
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
Extracted samples can be stored at -20 °C (or -80 °C) for further processing. Proceed to step 10.
?. [One-step RT and pre-amplification with Taqman assays]
Pool primer and probes :- Intermediate solution: mix together primers and probe for each target gene ( substep 10.1)- Pooled Taqman assay mix: pool intermediate solution (each assay) into a final pooled assay (substep 10.2)
?. [One-step RT and pre-amplification with Taqman assays]
Prepare One-step Reverse transcription and pre-amplification Pre-Mix :Components are from the Cells Direct One-Step qRT-PCR kit (Invitrogen, cat. no. 46-7200). In a DNA-free hood, prepare the following premix:  ABCDE1ComponentVolume for 1 reaction(µL)Vol. for 48 Reactions* (μL)Vol. for 96 Reactions* (μL)Vol. for 192 Reactions* (μL)2Pooled Taqman assay mix (step 10.2)6.2533066013203Superscript III RT/ Platinum Taq0.526.452.8105.642x Reaction mix12.5660132026405Water0.2513.226.452.8* Includes 10% overage.
ABCDE1ComponentVolume for 1 reaction(µL)Vol. for 48 Reactions* (μL)Vol. for 96 Reactions* (μL)Vol. for 192 Reactions* (μL)2Pooled Taqman assay mix (step 10.2)6.2533066013203Superscript III RT/ Platinum Taq0.526.452.8105.642x Reaction mix12.5660132026405Water0.2513.226.452.8
?. [One-step RT and pre-amplification with Taqman assays]
In a PCR plate, aliquot 19.5 μL of pre-mix for each sample.
?. [One-step RT and pre-amplification with Taqman assays]
Remove the plate from the DNA-free hood and add 5.5 μL of sample to each well containing pre-mix, making a total volume of 25 μL. If raw sample if used, this step should be performed in a BSL2 laboratory.
?. [One-step RT and pre-amplification with Taqman assays]
Mix the reactions by briefly vortexing, and then centrifuge
Centrifuge: 1000 33, 1 min
?. [One-step RT and pre-amplification with Taqman assays]
After cycling, dilute the reaction 1:5 by adding 100 μL Dilution Reagent (Fluidigm PN 100-8726) to the 25 μL reaction volume for a total volume of 125 μL.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Manufacturer's instructions for general 192.24 IFC protocolPrepare the IFCInject control line fluid into accumulator 2 (Acc2) on the IFC.Manufacturer's intruction for control line fluid loading
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Remove and discard the blue protective film from the bottom of the IFC.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Prepare 10X AssaysIn a DNA-free hood, prepare aliquots of 10X assays using volumes in the following table. Scale up appropriately for multiple runs.  ABCD1ComponentVol. per
  inlet (μL)Vol. per inlet with overage (μL)Vol. for 50 μL
  stock2Intermediate solution (from step 10.1)1.52.025.032X Assay Loading Reagent (Fluidigm PN 100-7611)1.52.025.04Total3.04.050.0
ABCD1ComponentVol. per
  inlet (μL)Vol. per inlet with overage (μL)Vol. for 50 μL
  stock2Intermediate solution (from step 10.1)1.52.025.032X Assay Loading Reagent (Fluidigm PN 100-7611)1.52.025.04Total3.04.050.0
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Prepare Sample Pre-Mix and SamplesCombine components in the following table to make sample pre-mix and final sample mixture. Scale up appropriately for multiple runs.  ABCD1ComponentVol. per
  inlet (μL)Vol. per inlet with overage (μL)Sample pre-mix for
  192.24 with overage* (μL)2TaqMan Universal PCR Master Mix (2X) (Life Technologies PN 4304437)1.52.0480.0320X GE Sample Loading Reagent (Fluidigm
PN 100-7610)0.150.248.0
ABCD1ComponentVol. per
  inlet (μL)Vol. per inlet with overage (μL)Sample pre-mix for
  192.24 with overage* (μL)2TaqMan Universal PCR Master Mix (2X) (Life Technologies PN 4304437)1.52.0480.0320X GE Sample Loading Reagent (Fluidigm
PN 100-7610)0.150.248.0
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
In a DNA-free hood, combine the TaqMan Universal PCR Master Mix with the GE Sample Loading Reagent in a 1.5 mL sterile tube—enough volume to fill an entire IFC. 2.2 μL of this sample pre-mix can then be aliquoted for each sample.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Remove these aliquots from the DNA-free hood and add 1.8 μL of cDNA to each, making a total volume of 4 μL in each aliquot.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Pipet 3 μL of each assay and 3 μL of each sample into the respective inlets on the IFC (see the 192.24 IFC pipettingmap).
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Pipet 150 μL of Actuation Fluid into the P1 well on the IFC.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Pipet 150 μL of Pressure Fluid into the P2 and P3 wells on the IFC.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Pipet 20 μL of Pressure Fluid into the P4 and P5 wells on the IFC.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Blot the carrier surface with a dry, lint-free cloth.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Start data collection on Biomark HD
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Start a New Run.Ensure that the status indicators for the lamp and the camera are green.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Place the loaded IFC into the Biomark HD.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose project settings (if applicable). Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Load.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Verify IFC barcode and IFC type.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose project settings (if applicable). Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Provide a name and select a file storage location for a new IFC run, or browse to select a predefined run file. Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose the application, reference, and probes: a Application type: Gene Expression b Passive reference: ROX c Assay: Single probe d Probe type: FAM-Non fluo or FAM-MGB e Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Browse to and choose the thermal protocol: below the thermal protocol (Taqman Covid) we have used:
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Confirm Auto Exposure is selected. Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Verify IFC run information
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Start Run
?. [RNA extraction with QIAamp Viral RNA Mini Kit Qiagen]
See manufacturer's instructions in the following file.Transfer 140 µL sample into a 1.5 mL microcentrifuge tube.
?. [Direct RTqPCR on sample]
If performing RT-qPCR without prior RNA extraction and purification, starting samples in Viral Transport Medium or in 10mM Tris-HCl pH 6.8 and 1mM EDTA, proceed to either of the following:- heat inactivate only (substep 9.1).- treat with QuickExtract DNA extraction DNA solution (from Lucigen) or 0,5% Tween 20 and 2mg/mL Proteinase K (substeps 9.2 and 9.3).
?. [Direct RTqPCR on sample]
If performing direct heat inactivation only:Transfer 25µl of sample in a 0.2 mL PCR-grade tube and incubate 5 min at 95°C in a thermo cycler.Then, keep the treated sample at +4°C until the One Step RT-PreAmp reaction (proceed to Step 10).
?. [Direct RTqPCR on sample]
If performing treatment using QuickExtract DNA extraction solution or Tween 20/Proteinase K:Prepare a 2X master mix solution for the Tween 20/Proteinase K treatment (using 2mg/mL proteinase K in 0,5% tween 20 as final conditions), as indicated in the following table:  ABC1ComponentsVolume (µL)Final concentration2100mM Tris HCl pH6.812020 mM350 mM EDTA242 mM420% Tween20301%510 mg/mL Proteinase K2404mg/mL6H2O1867final volume600In 0.2mL PCR-grade tubes, combine 25 µL of the 2X Tween 20/ProteinaseK master mix or 25 µL of QuickExtract DNA extraction solution with 2 5µL of the sample. Pipet up and down to mix well and spin down quickly.
ABC1ComponentsVolume (µL)Final concentration2100mM Tris HCl pH6.812020 mM350 mM EDTA242 mM420% Tween20301%510 mg/mL Proteinase K2404mg/mL6H2O1867final volume600
?. [Direct RTqPCR on sample]
Incubate the tube at 65°C for 10 min, 95°C for 5 min. Do not incubate tubes directly at +4°C but instead let the treated samples at room temperature (optional: you can store tube on ice afterwards).Samples are ready to use, proceed to step 10.
?. [One-step RT and pre-amplification with Taqman assays]
Prepare and intermediate solution by mixing primers and probe for each target, with the following volumes:  ABC1ComponentVolume (µL)Final concentration2Forward primer (100 µM)16.776.7 µM3Reverse primer (100 µM)16.776.7 µM4Probe (100 µM)4.251.7 µM5Water212.216Total Volume250
ABC1ComponentVolume (µL)Final concentration2Forward primer (100 µM)16.776.7 µM3Reverse primer (100 µM)16.776.7 µM4Probe (100 µM)4.251.7 µM5Water212.216Total Volume250
?. [One-step RT and pre-amplification with Taqman assays]
Pool each intermediate solution into a pooled Taqman assay mix (0,2X) using TE (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) so that each primer is at a final concentration of 180 nM, and each probe at 50 nM.This step should be adapted depending on the number of primers/probes to be used in the final pooled assay (ideally 8 or 12 sets of primers for a 192.24 IFC).The chart below provides an example using 4 assays used to detect SARS-CoV-2  AB1ComponentVolume (μL)2Intermediate solution (6.7/1.7 μM)6 (each assay) x 43TE1764Total200Note: Volume can be adjusted proportionally based on the number of samples to be amplified
AB1ComponentVolume (μL)2Intermediate solution (6.7/1.7 μM)6 (each assay) x 43TE1764Total200
?. [One-step RT and pre-amplification with Taqman assays]
Place in a standard thermal cycler.Incubate using the following protocol:  ABCD1ConditionTemperatureTime2Hold42 °C15 min3Hold95 °C2 min420 cyclesDenaturation95 °C15 s5Annealing60 °C2 min6Hold4 °C∞
ABCD1ConditionTemperatureTime2Hold42 °C15 min3Hold95 °C2 min420 cyclesDenaturation95 °C15 s5Annealing60 °C2 min6Hold4 °C∞
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Verify IFC barcode and IFC type.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose project settings (if applicable). Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Provide a name and select a file storage location for a new IFC run, or browse to select a predefined run file. Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose the application, reference, and probes: a) Application type: Gene Expression b) Passive reference: ROX c) Assay: Single probe d) Probe type: if using a dark quencher choose FAM-Non fluo (Need to be added manually into the Biomark) or FAM-MGB. e) Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Browse to and choose the thermal protocol: below the thermal protocol (Taqman Covid) we have used:
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Confirm Auto Exposure is selected. Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Verify IFC run information
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Start Run
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Prime IFC on the Controller.Place the IFC into the instrument and run the load script:- Juno:Load Mix 192.24 GE- RX: Load Mix (169x)IMPORTANT : Start IFC run within 1 hour of loading samples.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Remove any dust particles or debris from the IFC surface with clear tape.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Load.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Choose project settings (if applicable). Click Next.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Place the loaded IFC into the Biomark HD.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Click Start a New Run.Ensure that the status indicators for the lamp and the camera are green.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Double-click the Data Collection icon on the desktop.
?. [Gene Expression with the 192.24 IFC Using Standard TaqMan Assays]
Start data collection on Biomark HD