TITLE: IPMC SARS-CoV-2 Two-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]
?. [RNA Extraction with QIAamp Viral RNA Mini Kit Qiagen]
To be performed in the appropriate biosafety conditions (BSL2 laboratory)Transfer the totality of the transport medium into a 2 mL cryotube. OPTIONAL: in the case of a non-virucide transport medium, heat the sample for 10 min at 65°C. This step should strongly decrease the virus infectivity but might not end up in total inactivation. The effect of heat inactivation may lead to a decrease of test sensitivity depending on the type of swab transport medium.   Manufacturer's instructions in the attached file.OPTIONAL: Addition of carrier RNA (provided in the kit) to Buffer AVL to improve yield. Follow the table provided in the handbook to prepare a solution containing 5.6 μg of carrier RNA per sample. We have compared protocols containing or not this step and did not find any significant differences regarding the sensitivity of detection. Transfer 140 µL sample into a 1.5 mL microcentrifuge tube.
?. [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]
Briefly centrifuge the tube to remove drops from the inside of the lid
?. [RNA Extraction with QIAamp Viral RNA Mini Kit Qiagen]
Go to Qiacube.  Elution volume: default 60 µLSee Qiacube protocol in the attached file.
?. [cDNA Preparation with Reverse Transcription Master Mix]
Thaw all reagents on ice. Briefly vortex and centrifuge the reagents before using.Manufacturer's instructions in the attached file.
?. [cDNA Preparation with Reverse Transcription Master Mix]
In a PCR plate (on ice), prepare a pre-mix of the Reverse Transcription Master Mix and water as indicated in the following table.  ABCDE1  Component    <b style="mso-bidi-font-weight:
  normal">Volume per Reaction (μL)  <b style="mso-bidi-font-weight:
  normal">Volume for 48 Reactions* (μL)  <b style="mso-bidi-font-weight:
  normal">Volume for 96 Reactions* (μL)    <b style="mso-bidi-font-weight:
  normal">Volume for 192 Reactions* (μL)  2  Reverse Transcription
  Master Mix    1.0    52.8    105.6    211.2  3  RNase-free
  water    2.0    105.6    211.2    422.4  * Includes 10% overage for ease of pipetting.
ABCDE1  Component    <b style="mso-bidi-font-weight:
  normal">Volume per Reaction (μL)  <b style="mso-bidi-font-weight:
  normal">Volume for 48 Reactions* (μL)  <b style="mso-bidi-font-weight:
  normal">Volume for 96 Reactions* (μL)    <b style="mso-bidi-font-weight:
  normal">Volume for 192 Reactions* (μL)  2  Reverse Transcription
  Master Mix    1.0    52.8    105.6    211.2  3  RNase-free
  water    2.0    105.6    211.2    422.4
?. [cDNA Preparation with Reverse Transcription Master Mix]
Add 2 µL of RNA to each well containing pre-mix, to reach a total volume of 5 μL.
?. [cDNA Preparation with Reverse Transcription Master Mix]
Properly seal and gently vortex to mix the reverse transcription reactions.
?. [cDNA Preparation with Reverse Transcription Master Mix]
Centrifuge the reactions
Centrifuge: 1000 33, 1 min
?. [cDNA Preparation with Reverse Transcription Master Mix]
Place in a standard thermal cycler.Incubate using the following protocol:  ABC1ConditionTemperatureTime2Hold25 °C5 min3Hold42 °C30 min4Hold85 °C5 min5Hold4 °C∞
ABC1ConditionTemperatureTime2Hold25 °C5 min3Hold42 °C30 min4Hold85 °C5 min5Hold4 °C∞
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Prepare Pre-Mix and Samples:In a DNA-free hood, prepare the sample pre-mix for the reactions as shown in the following table:  ABCDE1ComponentVol. per Reaction (μL)Vol. for 48 Reactions* (μL)Vol. for 96 Reactions* (μL)Vol. for 192 Reactions* (μL)2Preamp Master Mix (Fluidigm PN 100-5744)1.0052.8105.6211.23Pooled TaqMan assay mix (0.2X)1.2566.0132.0264.04Water----* Includes 10% overage.
ABCDE1ComponentVol. per Reaction (μL)Vol. for 48 Reactions* (μL)Vol. for 96 Reactions* (μL)Vol. for 192 Reactions* (μL)2Preamp Master Mix (Fluidigm PN 100-5744)1.0052.8105.6211.23Pooled TaqMan assay mix (0.2X)1.2566.0132.0264.04Water----
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
In a PCR plate, aliquot 2.25 μL of pre-mix for each sample.
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Remove the plate from the DNA-free hood and add 2.75 μL of cDNA to each well containing pre-mix, making a total volume of 5 μL.
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Mix the reactions by briefly vortexing, and then centrifuge
Centrifuge: 1000 33, 1 min
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Place the plate in the thermal cycler and cycle using the following table as a guide:  ABCD1ConditionTemperatureTime2Hold           95 °C2 min320 cyclesDenaturation95 °C15 s4Annealing/extension60 °C2 min
ABCD1ConditionTemperatureTime2Hold           95 °C2 min320 cyclesDenaturation95 °C15 s4Annealing/extension60 °C2 min
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
After cycling, dilute the reaction 1:5 by adding 20 μL Dilution Reagent (Fluidigm PN 100-8726) to the final 5 μL reaction volume for a total volume of 25 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 (step 14.1)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 (step 14.1)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).
?. [RNA Extraction with QIAamp Viral RNA Mini Kit Qiagen]
lncubate for 10 min
0 Room temperature
?. [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]
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]
Remove any dust particles or debris from the IFC surface with clear tape.
?. [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]
Double-click the Data Collection icon on the desktop.
?. [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: 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
?. [RNA Extraction with QIAamp Viral RNA Mini Kit Qiagen]
Extracted samples can be stored at -20 °C (or -80 °C) for further processing.
?. [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.
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Pool primer and probes with 2 steps:- Intermediate solution: mix together primers and probe for each target gene (substep 14.1)- Pooled Taqman assay mix: pool the intermediate solution into a final pooled assay (substep 14.2)
?. [Fluidigm Preamp Master Mix and TaqMan Assays]
Prepare an 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
?. [Fluidigm Preamp Master Mix and 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