TITLE: Semi-Automated and Miniaturized SARS-CoV-2 Detection using TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay
AUTHORS: Peter De Hoff, Sydney Morgan, Louise  C Laurent

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this procedure is to describe the setup of a semi-automated and miniaturized workflow for SARS-CoV-2 detection using the TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay.</div><div class = "text-block"><span>This procedure applies to specimens received for SARS-CoV-2 testing in a clinical setting. Samples are collected, processed, and RNA is extracted as described elsewhere, using the </span><a href="https://www.protocols.io/edit/semi-automated-extraction-of-viral-rna-using-the-m-brcvm2w6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">MagMax Viral Pathogen (MVPII) 96 Kit for SARS-CoV-2 Detection</span></a><span> protocol or the </span><a href="https://www.protocols.io/edit/semi-automated-extraction-of-viral-rna-using-the-o-brqhm5t6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Omega Bio-tek Mag-Bind Viral DNA/RNA 96 Kit for SARS-CoV-2 Detection</span></a><span> protocol. The TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay is a multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19). The procedure has been modified to accommodate </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span>  reactions as opposed to the standard </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span> reactions, significantly reducing per-reaction cost. This is achieved by using the SPT LabTech Mosquito HV and the SPT LabTech Mosquito X1 liquid handling robots.</span></div><div class = "text-block">Purified viral ribonucleic acid (RNA) is reverse-transcribed into cDNA and amplified using the TaqPath™ COVID-19 Combo Kit and the QuantStudio 7 Pro 384 Real-Time PCR Systems. In the process, the probe oligos anneal to four (4) specific target sequences located between four (4) unique forward and reverse primers for the following genes: ORF1ab, N Gene, S Gene, and the MS2 bacteriophage RNA extraction positive control. During the extension phase of the qPCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each qPCR cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored during each PCR cycle by the QuantStudio 7 Pro 384 Real-Time PCR System.</div></div>

[STEPS]
?. [Workstation Preparation]
Set up:Turn on equipment (Mosquito HV, Mosquito X1, QuantStudio 7, Centrifuge) and ensure all equipment and programs start up correctly.    Ensure the centrifuge is set to   Decontaminate workspace, pipettes, and equipment using RNASEZap or equivalent.Fill two (2) ice trays with crushed ice.Ensure the centrifuge is set to Decontaminate workspace, pipettes, and equipment using RNASEZap or equivalent.Fill two (2) ice trays with crushed ice.For rapid thawing of frozen reagents, add 2-3cm deep worth of  to a third ice tray, then add crushed ice (enough so ice is still floating) and a floating tube holder into the water.
4 °C
0 Room temperature
?. [Assay Controls]
Internal Control:The MS2 Phage Internal Control is used to monitor RNA extraction and was added at the RNA extraction step - see related RNA-extraction protocols using the MagMax Viral Pathogen (MVPII) kit or the Omega Bio-tek Mag-Bind Viral DNA/RNA kit. The MS2 Phage Internal Control is added to every sample/well of the reaction plate except for the Positive Control well(s).Negative Control:The Negative Control (nuclease-free water with MS2 Phage Internal Control) is used to monitor efficiency of RNA extraction process. One Negative Control is included in every 96-well block (i.e. one Negative control per 94 samples) and is added after patient and mock material is added to the RNA extraction reagent during the RNA extraction process.Positive Control:The Positive Control (TaqPathTM COVID-19 Control) is used to validate the operation of ther RT-qPCR reaction process. One Positive Control is included in every 96-well block (i.e. one Positive Control per 94 samples) and is added at the RT-qPCR reaction step.Positive Control:*For data analysis and results interpretation, refer to Step 9 below: Data Analysis and Interpretation.
?. [Sample Preparation]
If not proceeding directly from the Semi-Automated Extraction of Viral RNA using the MagMax Viral Pathogen (MVPII) 96 Kit for SARS-CoV-2 Detection RNA extraction protocol OR the Semi-Automated Extraction of Viral RNA using the Omega Bio-tek Mag-Bind Viral DNA/RNA 96 Kit for SARS-CoV-2 Detection protocol, locate and remove the appropriate 384-well RNA Working Plates (RWPs) from their storage location (ultra-low freezer). Place on a 384-well aluminum block at  until thawed.Once thawed, place RWP on a prechilled aluminum block and keep  .
0 Room temperature
on ice
?. [Master Mix Preparation]
Prepare the Master Mix (MM):Keep MM tube  when creating the master mix. Once all reagents have been added, close the lid, vortex gently to mix, and centrifuge briefly to collect all liquid at the bottom of the tube.ReagentsPer 1 reactionFor 96 reactions (+ error)For 192 reactions (+ error)For 288 reactions (+ error)For 384 reactions (+ error)Nuclease-Free Water0.10 µL13.6 µL23.5 µL33.5 µL43.5 µLProbe Mix0.15 µL20.4 µL35.2 µL50.2 µL65.2 µL4x Single Step RT-qPCR Enzyme Mix0.75 µL101.8 µL176.0 µL251.0 µL326.0 µLMM Total:1.0 µL136.5 µL236.4 µL337.2 µL438.1 µLMM Volume to add to each well of IMP:N/A8.2 µL14.2 µL20.3 µL26.3 µL
This process involves making a qPCR Master Mix (MM) in a 1.5 mL microcentrifuge tube, then aliquoting this mixture into the Intermediate Master Mix Plate (IMP) by hand, before it is transferred to the qPCR Working Plate (QRP) using the Mosquito HV (Step 5 below). Samples will ideally be organized in multiples of 96 (94 samples + Positive and Negative Controls).
Preparation of the MM and the IMP should ideally be performed in a dead air PCR hood that has been prepared for work with RNA.
on ice
ReagentsPer 1 reactionFor 96 reactions (+ error)For 192 reactions (+ error)For 288 reactions (+ error)For 384 reactions (+ error)Nuclease-Free Water0.10 µL13.6 µL23.5 µL33.5 µL43.5 µLProbe Mix0.15 µL20.4 µL35.2 µL50.2 µL65.2 µL4x Single Step RT-qPCR Enzyme Mix0.75 µL101.8 µL176.0 µL251.0 µL326.0 µLMM Total:1.0 µL136.5 µL236.4 µL337.2 µL438.1 µLMM Volume to add to each well of IMP:N/A8.2 µL14.2 µL20.3 µL26.3 µL
?. [Transfer Master Mix to qPCR Reaction Plate]
Set up Mosquito HV Computer:16 reactions/one column: 384 reactions/full plate:
This process transfers the MM from the Intermediate Master Mix Plate (IMP) to the qPCR Reaction Plate (QRP) using the Mosquito HV. To each well of the QRP, 1 µL MM is added.
?. [Positive Control Addition to qPCR Reaction Plate]
Prepare the Positive Control Plate (PCP):Place one aliquot of diluted RT-qPCR Positive Control RNA in a tube holder floating in ice water until thawed, then place  .Add an aliquot of the Positive Control to an empty well of the Positive Control Plate (PCP): add  Positive Control RNA for each Positive Control reaction on the QRP + 2uL of this RNA for instrument dead volume. (2.5uL total control RNA for a single well, 4uL total for four wells.)
The result of this process is the addition of the COVID-19 Positive Control viral RNA material into the appropriate wells of the QRP using the Mosquito X1. One Positive Control is included for each block of 96 reactions (i.e. one Positive Control per 94 samples). If RNA was extracted using the MagMax protocol, the Positive Controls are placed in wells B2, B8, B14, and B20 of the 384-well QRP.If RNA was extracted using Omega protocol, the Positive Controls are placed in wells A1, A7, A13, and A19 of the 384-well QRP.*One (1) Positive Control must be added for each block of 96 samples, or portion thereof. Therefore, 10 samples require one (1) Positive Control, and 100 samples require two (2) Positive Controls.*
on ice
0.5 µl
?. [RNA Addition to qPCR Reaction Plate]
Prepare RNA Working Plate (RWP):At this time, RWP is thawed, and sitting   (see Step 3 above).Centrifuge Place a 384-well magnetic plate onto a 384-well aluminum plate   to chill the magnetic plate without it getting wet.
In this process,   aliquots of viral RNA are transferred from the RWP to the QRP using the Mosquito HV.
on ice
Centrifuge: 2200 34, 3 min, 4 10
on ice
on ice
?. [Perform RT-qPCR]
Set up the QuantStudio 7 (or QuantStudio 5) and Run:Turn on the QuantStudio 7 Pro (or QuantStudio 5) qPCR machineEnter the reaction plate barcode into the "Filename" portion.Insert the plate into the machine, ensuring well A1 is in the upper left corner.Run the following RT-qPCR program (Table 4):Cycle StepTemperatureTimeCyclesIncubation25 C2 min1Reverse Transcription53 C10 min1Activation95 C2 min1Denaturation95 C3 sec55Anneal/Extension60 C30 secHold4 Cholding1
Cycle StepTemperatureTimeCyclesIncubation25 C2 min1Reverse Transcription53 C10 min1Activation95 C2 min1Denaturation95 C3 sec55Anneal/Extension60 C30 secHold4 Cholding1
?. [Data Analysis and Interpretation]
Results interpretation: ORF1ab, N gene, and S gene targetsCq CutoffCq ConfidenceTarget Call> 0.7Positive (+)Negative (-)> 37Negative (-)> 37> 0.7Negative (-)Results interpretation: MS2 Internal Positive Control Cq CutoffCq ConfidenceTarget Call> 0.3Positive (+)Negative (-)> 37Negative (-)> 37> 0.3Negative (-)
The purpose of this step is to analyze, interpret, and report the results of the RT-qPCR assay for the detection of SARS-CoV-2.The individual gene target calls/results interpretations (Tables 5 and 6) are used in the subsequent steps to provide assessment calls for each sample. A target call of "Positive" signifies that the gene has successfully amplified. A target call of "Negative" signifies that there is no signal of gene amplification.
Cq CutoffCq ConfidenceTarget Call> 0.7Positive (+)Negative (-)> 37Negative (-)> 37> 0.7Negative (-)
Cq CutoffCq ConfidenceTarget Call> 0.3Positive (+)Negative (-)> 37Negative (-)> 37> 0.3Negative (-)
?. [Workstation Preparation]
Consumable Preparation:Scan the barcodes for the following supplies (Table 1) into the INSPECT application:ItemBarcodeConsumable# Needed Per RunRNA Working Plate (RWP)######Hard-Shell 384-Well PCR Plate1qPCR Reaction Plate (QRP)######Hard-Shell 384-Well PCR Plate1Master Mix (MM) TubeNo Barcode1.5 mL Microcentrifuge Tube1Intermediate MM Plate######Hard-Shell 384-Well PCR Plate (one column used per run, plate re-useable up to 24 times – one time for each column)1Positive Control Plate######Hard-Shell 384-Well PCR Plate (re-useable)1
ItemBarcodeConsumable# Needed Per RunRNA Working Plate (RWP)######Hard-Shell 384-Well PCR Plate1qPCR Reaction Plate (QRP)######Hard-Shell 384-Well PCR Plate1Master Mix (MM) TubeNo Barcode1.5 mL Microcentrifuge Tube1Intermediate MM Plate######Hard-Shell 384-Well PCR Plate (one column used per run, plate re-useable up to 24 times – one time for each column)1Positive Control Plate######Hard-Shell 384-Well PCR Plate (re-useable)1
Steps involving the INSPECT application are optional and are not essential for performing RT-qPCR detection of SARS-CoV-2 viral RNA.
?. [Workstation Preparation]
Reagent Preparation:Reagent NameQuantityStorage ContainerStorage ConditionsHandling ConditionsTaqPath™ 1-Step Multiplex Master Mix 12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.COVID-19 Real Time PCR Assay Multiplex12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.  Cover at all times to protect from light.TaqPathTM COVID-19 Control Dilution Buffer12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.Positive Control: TaqPathTM COVID-19 Control11 mL tube-80C freezerThaw in ice water tray and then transfer to other ice tray.Nuclease-free water1100 mL ContainerBenchtopKeep at RT
Reagent NameQuantityStorage ContainerStorage ConditionsHandling ConditionsTaqPath™ 1-Step Multiplex Master Mix 12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.COVID-19 Real Time PCR Assay Multiplex12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.  Cover at all times to protect from light.TaqPathTM COVID-19 Control Dilution Buffer12 mL tube-20C freezerThaw in ice water tray and then transfer to other ice tray.Positive Control: TaqPathTM COVID-19 Control11 mL tube-80C freezerThaw in ice water tray and then transfer to other ice tray.Nuclease-free water1100 mL ContainerBenchtopKeep at RT
?. [Master Mix Preparation]
Transfer MM to IMP:Place the IMP on a chilled 384-well aluminum block and keep  .Mark the position of an unused column on the IMP, and to each well of that column, add the appropriate amount of master mix based on the last row of Table 3 above.Seal the IMP with a temporary plate seal and centrifuge  . Check that each well has an equal volume of liquid, and double check that there are not bubbles in any well, which could interfere with reagent transfer.Seal the IMP with a temporary plate seal and centrifuge . Place the IMP on a 384-well aluminum block  and cover to shield from light.
on ice
Centrifuge: 2000 34, 30 s, 4 10
on ice
If there are bubbles, centrifuge again, coarsely flick the well(s) that contain bubbles, or pop the bubbles with a fine pipette tip.
?. [Transfer Master Mix to qPCR Reaction Plate]
Set up Mosquito HV Plate Deck:On the Mosquito HV Computer, select the appropriate source column from the IMP source plate (the column containing the MM) and begin the program.Push the “Move Left” button on the computer and the plate deck will move to the left, allowing access to Positions 2 and 3 on the plate deck.Place the QRP in Position 2 of the plate deck.Place the IMP in Position 3 of the plate deck.
When adding the plates to the Mosquito HV plate deck, ensure the lower right corner of each plate is resting in front of, and not on top of, the diagonal (spring-loaded) plate securing bar.
?. [Transfer Master Mix to qPCR Reaction Plate]
Run the Program:When the plates have been loaded onto the plate deck and the program is correct, select "Run" on the Mosquito HV computer. After the run is complete, remove the QRP, tap the plate gently to drive large drops towards the bottom of the wells, seal with a temporary plate seal, and centrifuge   to move all liquid to the bottom of each well.Hold the plate to the light and verify that all wells have an equal volume of liquid.After the run is complete, remove the QRP, tap the plate gently to drive large drops towards the bottom of the wells, seal with a temporary plate seal, and centrifuge  to move all liquid to the bottom of each well.
Observe the pipette tips in action: if the tips are not entering the wells properly, hit the "Pause" or "Abort" buttons and troubleshoot (troubleshooting not included in this protocol).
Centrifuge: 2000 34, 30 s, 4 10
If there are wells without any MM, manually add with a P2 pipette.
?. [Transfer Master Mix to qPCR Reaction Plate]
Post-Run:Store the QRP   and cover to shield from light.
on ice
Only one column of the IMP is used at a time: the IMP can be re-used until all columns have been used before being discarded.
?. [Positive Control Addition to qPCR Reaction Plate]
Set up Mosquito X1 Computer:Addition of one Positive Control: Addition of four Positive Controls:
Ensure that the X1 program properly references the correct source well on the PCP.
?. [Positive Control Addition to qPCR Reaction Plate]
Set up Mosquito X1 Plate Deck:Place the PCP in Position 1 (left position) of the X1 plate deck.Remove the temporary seal from the QRP and place the plate in Position 2 (right position) of the X1 plate deck.
When adding the plates to the Mosquito X1 plate deck, ensure the lower right corner of each plate is resting in front of, and not on top of, the diagonal (spring-loaded) plate securing bar.
?. [RNA Addition to qPCR Reaction Plate]
Set up the Mosquito HV Computer:For 16 reactions/one column: For 384 reactions/full plate:
?. [Positive Control Addition to qPCR Reaction Plate]
Run the Program:When everything is correctly positioned, select "Run". The X1 will add   of the COVID-19 Positive Control to the appropriate well(s) of the QRP.When the run has finished, remove the QRP, seal with a temporary plate seal, and centrifuge . Check the appropriate wells for volume change to confirm the Positive Control was added.Place QRP on the 384-well aluminum block   and cover to shield from light.
0.5 µl
Centrifuge: 2000 34, 30 s, 4 10
on ice
Mark the used column of the PCP with an "X" so it is not accidentally used again - can re-use this plate up to 24 separate times (once per column on the plate).
?. [RNA Addition to qPCR Reaction Plate]
Run the Program:When everything is correctly positioned, select "Run".When the run is complete, remove the RWP and seal completely with a Bio-Rad Microseal 'B' plate seal. Store at   .Remove the QRP and seal completely with a MicroAmp Optical Adhesive Film plate seal. Properly center the plate seal on the plate, and seal completely around the edges and around each well, using a speedball soft rubber roller and a plastic wedge sealer.Centrifuge the QRP    and ensure there are no bubbles in any of the wells.Transfer the QRP to a chilled 384-well aluminum block on ice and transport to the QuantStudio 7 PCR Machine.When the run is complete, remove the RWP and seal completely with a Bio-Rad Microseal 'B' plate seal. Store at  .Remove the QRP and seal completely with a MicroAmp Optical Adhesive Film plate seal. Properly center the plate seal on the plate, and seal completely around the edges and around each well, using a speedball soft rubber roller and a plastic wedge sealer.Centrifuge the QRP    and ensure there are no bubbles in any of the wells.Transfer the QRP to a chilled 384-well aluminum block on ice and transport to the QuantStudio 7 PCR Machine.Centrifuge the QRP   and ensure there are no bubbles in any of the wells.
-80 °C
Centrifuge: 2200 34, 1 min, 4 10
When sealing the QRP, be careful to ensure no wrinkles are present in the plate seal, as miniaturized reactions can have a very small surface area for fluorescence, and thus any distortions in the light path can dramatically impact performance.
?. [RNA Addition to qPCR Reaction Plate]
Set up the Mosquito HV Plate Deck:Remove the seals from the RWP and QRP.Place the RWP (and the magnetic block it sits on) onto Position 4 of the Mosquito HV plate deck. Ensure the plate is properly oriented, with will A1 in the upper left corner.Place the QRP onto Position 2 of the Mosquito HV plate deck, ensuring the plate is properly oriented.
The empty deck position between the RWP and the QRP is used for tip changing and reduces the potential for cross contamination of samples.
?. [Data Analysis and Interpretation]
Assessment calls: ControlsControl SampleORF1abN GeneS GeneMS2 Internal ControlAssessment CallNegative Control---+Negative (-)Positive Control+++-Positive (+)
Control SampleORF1abN GeneS GeneMS2 Internal ControlAssessment CallNegative Control---+Negative (-)Positive Control+++-Positive (+)
?. [Data Analysis and Interpretation]
Assessment calls: SamplesORF1abN GeneS GeneMS2 Internal ControlAssessment Call+++-Positive (+)-++-Positive (+)+-+-Positive (+)++--Positive (+)+---Inconclusive-+--Inconclusive--+-Inconclusive---+Negative (-)----Invalid
For overall sample assessment:Positive result: At least 2/3 gene targets (ORF1ab, N gene, S gene) are positive (MS2 internal control can be positive or negative)Inconclusive result: 1/3 gene targets are positive (MS2 internal control can be positive or negative)Negative result: 0/3 gene targets are positive + MS2 internal control is positiveInvalid result: 0/3 gene targets are positive + MS2 internal control is negativeIf a sample is Inconclusive or Invalid, re-extract RNA and re-perform RT-qPCR.
ORF1abN GeneS GeneMS2 Internal ControlAssessment Call+++-Positive (+)-++-Positive (+)+-+-Positive (+)++--Positive (+)+---Inconclusive-+--Inconclusive--+-Inconclusive---+Negative (-)----Invalid
?. [Validation of Reaction Miniaturization]
This step describes the validation of the miniaturization of the testing kit that was granted Emergency Use Authorization by the U.S. Food and Drug Administration (FDA). This step does not need to be repeated.
?. As this is a 1uL dry dispense, the Mosquito HV Genomics requires accurate tip spool calibration and accurate plate definition parameters.