TITLE: Direct-lysis Saliva SARS-CoV-2 Detection 
AUTHORS: Suzie  Alarcon, Hannah  Dose,   Arturo Hernandez,   Kyle Tanguay, Stephen Wilson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A One-Step RT-qPCR assay based on the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel has been developed which can effectively detect SARS-CoV-2 particles from saliva samples which undergo both heat inactivation and direct lysis with detergent, eliminating the need to extract RNA</div><div class = "text-block">Assay has been automated on the Beckman BiomekFXpliquid handler</div><div class = "text-block">Heat inactivation of virus in saliva does not impact assay results and reduces overall risk</div><div class = "text-block">6ul Low Volume reaction allows for high-throughput screening at reduced cost</div></div>

[STEPS]
?. [Saliva Collection]
Prepare saliva collection packetsInclude:Large RNase, DNase-free tube into which participant can deposit saliva2ml Safe-Lock Eppendorf tube containing 1.5 ml TE pH 82ml screw-cap gasketed cryo-safe tubeTransfer pipette (plastic bulb type)Barcodes for tubes, or human readable labelsAlcohol wipesBiohazard specimen transport bag
?. [Saliva Collection]
Instruct participant to collect saliva and create Working DilutionInstructions to participant:Ideally, complete the saliva collection before your first meal and within one hour of drop-off. Saliva collection may be aided by being well-hydrated.If you have eaten, brushed your teeth, smoked, or chewed gum, please wait at least one hour before collecting your saliva. Refrain from using mouth wash for 12 hours before collecting your saliva.Wash hands thoroughly.Open clear bag and retrieve the packet containing the large collection tube.  Open the large collection tube and passively drool into the large collection tube until 2 mls of saliva have been collected. This is best done by allowing saliva to pool in the mouth, then tip your head forward and allow saliva to enter the tube.Passive saliva collection, as opposed to stimulated and expectorated saliva is preferred and samples collected passively may contribute to better detection of CoV particles. Any samples that appear viscous or containing mucus will be rejected and will not be tested.Remove some of the saliva with the transfer pipette and place into the small collection tube (MUST BE ALMOST FULL) AND into the working tube (MUST BE FULL) with the saliva you just collectedClose tubes and wipe both with the alcohol pads - thank you for helping to protect the researchers running this screening!Make sure to close both tubes tightly! Any tubes that come partially opened, leaking, or otherwise not tightly sealed will be rejected and not tested. Affix barcode to tubes and place back into biohazard bag. Dispose of large tube and transfer pipette11. Close bag12. Collect bag from participant and place on ice.
?. [Saliva Collection]
Prepare Saliva SamplesHeat-inactivate samples at 98C x 5 minutes or 65C x 30 minutes in a dry heat block with appropriate adapters. Allow tubes to cool Centrifuge 25% working dilution for 20 minutes at 300gLyse 25% working dilution with 30% Triton-X:Add 5 parts 25% saliva dilution to 1 part 30% Triton-X (example, 50ul saliva to 10ul 30% Triton-X)Mix by slowly pipetting. Triton is a foaming detergent, take care not to foam.Incubate for 5 minutes at room temperature. Add 5 parts 25% saliva dilution to 1 part 30% Triton-X (example, 50ul saliva to 10ul 30% Triton-X)Mix by slowly pipetting. Triton is a foaming detergent, take care not to foam.Incubate for 5 minutes at room temperature
?. [Saliva Collection]
Prepare Plasmid dilution. Plasmid stock is 200,000 copies/ul. Prepare 200 copies/ul dilution and treat as sample. (Example 50 ul of 200 copies/ul is lysed with 10 ul 30% Triton X, as with samples). Aliquot in separate clean space to mitigate chance of false positives.
?. [Prepare Master Mix]
Primer and Probe PreparationIf received lab-ready at 100uM, skip to dilution and aliquoting:  Upon receipt, store dried primers and probes at 2-8°C.  Precautions: These reagents should only be handled in a clean area and stored at appropriate temperatures (see below) in the dark. Freeze-thaw cycles should be avoided. Maintain cold when thawed.  Using aseptic technique, suspend dried reagents in nuclease-free water to 100uM and allow to rehydrate for 15 min at room temperature in the dark.  Mix gently and aliquot primers/probe in 300 μL volumes into pre-labeled tubes. Store a single, working aliquot of primers/probes at 2-8C in the dark. Store remaining aliquots at ≤ - 20C in a non-frost-free freezer. Do not refreeze thawed aliquots (stable for up to 4 months at 2-80C). Prepare working dilutions as follows:Primers: 20uMProbes: 5uM  8. Store aliquots of working dilutions at -20C in the dark in black microcentrifuge tubes.
?. [Prepare Master Mix]
Prepare Master Mix (MM), working on ice  ABCDE1Componentul x1[Stock] uM[Final] nMReporter Dye2N1 Fw0.15205003N1 Rv0.15205004N1 Probe0.155125FAM5N2 Fw0.15205006N2 Rv0.15205007N2 Probe0.155125SUN8RP Fw0.15205009RP Rv0.152050010RP Probe0.155125ATTO 64711TaqPath or UltraPlex Tough Mix1.54X1X12H2O0.1513Total6
ABCDE1Componentul x1[Stock] uM[Final] nMReporter Dye2N1 Fw0.15205003N1 Rv0.15205004N1 Probe0.155125FAM5N2 Fw0.15205006N2 Rv0.15205007N2 Probe0.155125SUN8RP Fw0.15205009RP Rv0.152050010RP Probe0.155125ATTO 64711TaqPath or UltraPlex Tough Mix1.54X1X12H2O0.1513Total6
?. [Prepare Master Mix]
Distribute 3 or 6.5ul (see below) MM to clean 384 well optical PCR plate (internally validate sensitivity of white vs clear plate) Mix 3ul lysed saliva sample with 3ul MM in plateORIf adding technical replicates, mix 6.5ul lysed saiva sample with 6.5ul MMand dispense 6ul from well containing 13ul of lysed saliva+MM to empty well
?. [Prepare Master Mix]
Visually inspect plate to ensure all expected wells contain reagent. Seal plate with optically clear film and centrifuge for 1 minute at 1200 RPM
?. [qPCR and Data Export]
Run qPCR according to software and manufacturer instructions. Ensure all probe fluorophores are selected.Step 4 can be 3-5 seconds  ABCD1StepDeg, CTimePurpose21252 minUNG Incubation325015 minT Incubation43952 minEnzyme Activation54955 secAmplification655630 secAmplification/ Read77Go to step 4 45 times
ABCD1StepDeg, CTimePurpose21252 minUNG Incubation325015 minT Incubation43952 minEnzyme Activation54955 secAmplification655630 secAmplification/ Read77Go to step 4 45 times
?. [qPCR and Data Export]
Export data. If cycler has background dye like ROX, account for that in machine's settings.
?. [Evaluate and Report]
Evaluate Data: Where "+" is Ct 40 or not a number (NaN), Expected batch control = "+" for both N1 and N2 for plasmid and "-" for NTCIn the case of Positive or Inconclusive, repeat sample with fresh 25% dilution from stock tube, and with a 12.5% dilution using MM as listed above. Add an additional reaction with RdRP_SARSr_P2 Probe and primers at following concentrations to rule out plasmid contamination/false positive. Multiplex with RNAseP primer and probe set as internal control.Component[Final] nMReporter DyeRdRP_SARSr_F2 Forward Primer,600-	RdRP_SARSr_R1 Reverse Primer800-RdRP_SARSr_P2 Probe100FAM (can order with SUN)If any probe produces artifacts in the plasmid control, reduce primer and probe concentrations by 15%. Validate on positive samples. Evaluate NTCs manually to inspect for sigmoidal curve - any sigmoidal curve in the NTC wells indicate possible contamination and plate should be repeated.   ABCDEFGH1N1N2RPPlasmid Positive ControlNTCTest TypeResultNext Step2++++-InitialPositiveRe-Test3++++-ConfirmatoryPositiveRelease Data4+-++-InitialPositiveRe-Test5++++-ConfirmatoryPositiveRelease Data6-+++-InitialPositiveRe-Test7++++-ConfirmatoryPositiveRelease Data8--++-ConfirmatoryInconclusiveRe-collect sample9--++-InitialNegativeRelease Data10---+-InitialSample FailRe-collect sample11anyanyanyIf any batch control returns unexpected result, assay failAnyAssay FailRe-run plate
Component[Final] nMReporter DyeRdRP_SARSr_F2 Forward Primer,600-	RdRP_SARSr_R1 Reverse Primer800-RdRP_SARSr_P2 Probe100FAM (can order with SUN)
ABCDEFGH1N1N2RPPlasmid Positive ControlNTCTest TypeResultNext Step2++++-InitialPositiveRe-Test3++++-ConfirmatoryPositiveRelease Data4+-++-InitialPositiveRe-Test5++++-ConfirmatoryPositiveRelease Data6-+++-InitialPositiveRe-Test7++++-ConfirmatoryPositiveRelease Data8--++-ConfirmatoryInconclusiveRe-collect sample9--++-InitialNegativeRelease Data10---+-InitialSample FailRe-collect sample11anyanyanyIf any batch control returns unexpected result, assay failAnyAssay FailRe-run plate
?. [Decontamination]
Complete thorough decontamination of all working areas as designated by local, federal, and or institutional guidelines.Submerge any racks or holders in quaternary ammonium at appropriate concentration for time recommended by manufacturer. Do not use quaternary ammonium near NGS equipmentFollow with 70 Ethanol decontaminationAny non-submergable equipment should be sprayed with quaternary ammonium and allowed a 20 minute contact time, followed by 70% EtOHAll residual saliva samples, if not being stored (-80C storage recommended), should be aspirated into a bleach solution with a final concentration of 10%.Dispose of all consumables and PPE according to local, federal, and or institutional guidelines.