TITLE: SalivaDirect™: RNA extraction-free SARS-CoV-2 diagnostics
AUTHORS: Chantal Vogels, Orchid Allicock, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.</div><div class = "text-block">Version 2 includes: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Optimized thermocycler conditions</li><li style = "counter-reset:ol0;list-style-type:disc;">Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers</li><li style = "counter-reset:ol0;list-style-type:disc;">Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Version 3 has been updated to remove steps for sample self-collection.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers. </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Version 6 includes:</li><li style = "counter-reset:ol0;list-style-type:disc;">Introducing multiple workflows for sample processing including heat Pre-treatment  as an alternative to Proteinase K </li><li style = "counter-reset:ol0;list-style-type:disc;">Updated list of RT-qPCR reagents, theromcyclers and protocol for 384 well format.</li></ul></div><div class = "text-block">Version 3 has been updated to remove steps for sample self-collection.</div><div class = "text-block">Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.</div><div class = "text-block">Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers. </div><div class = "text-block">Version 6 includes:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Introducing multiple workflows for sample processing including heat Pre-treatment  as an alternative to Proteinase K </li><li style = "counter-reset:ol0;list-style-type:disc;">Updated list of RT-qPCR reagents, theromcyclers and protocol for 384 well format.</li></ul></div></div>

[STEPS]
?. [Sample collection]
Saliva should be collected with the assistance of a healthcare worker or technician.
?. [Sample collection]
Before collection, clean hands using alcohol-based sanitizer or soap and water (no fragrances) and don appropriate PPE (at minimum, gloves and a mask).
?. [Sample collection]
Ensure all collection materials are labelled with the correct identifying information.
?. [Sample collection]
While preparing collection materials, direct the sample provider to begin pooling saliva in their mouth. Saliva production can be stimulated by thinking about food (favorite foods, upcoming meals, etc.) or about the saliva collection itself.
This protocol is intended for the collection of the normal saliva that naturally pools into the mouth. No coughing or sniffing prior to sample collection is required.Ideally, water should be avoided 10 minutes prior to collection. Other drinks, food, and nasal sprays should be avoided for half an hour before sample collection.
?. [Sample collection]
Remove the lid of the collection container, and direct the sample provider to gently expel saliva into the container until at least 0.5 mL has been collected.
The total volume measured is to exclude any bubbles.
?. [Sample collection]
Once at least 0.5 mL has been collected, securely replace the lid of the collection container.
?. [Sample collection]
Sterilize the container surface with 70% ethanol or a disinfecting wipe, and place the sample in a secondary container or an appropriately labeled biohazard bag.
?. [Sample collection]
Dispose of gloves, and register the sample collection (including date and time).
?. [Sample pretreatment]
Samples can either be treated with either:Workflow 1: Standard SalivaDirect protocolWorkflow 2: Pre-Treatment Heat step prior to SalivaDirect protocol without the addition of Proteinase K and heat inactivation stepWorkflow 3: Pre-Treatment Heat step prior to standard SalivaDirect protocol with Proteinase K and heat inactivation stepSelect the appropriate option below.
?. [RT-qPCR]
Prepare 20 uM working stocks of the primers and probes (sequences provided in Materials) by adding 20 µL of 100 uM stock to 80 µL nuclease-free water.
Briefly vortex and centrifuge reagents before use.Probes are photosensitive and should be stored in the dark.
?. [RT-qPCR]
Use the 20 uM working stocks to prepare dualplex primer-probe-water mix containing the following:  ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
?. [RT-qPCR]
Prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting), excluding processed saliva samples or control  Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath   Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -   Primer-probe- water mix (see Step 11.1)    4 µL    4 µL    4 µL   Nuclease-free water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath   Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -   Primer-probe- water mix (see Step 11.1)    4 µL    4 µL    4 µL   Nuclease-free water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
For the ABI 7500 Fast Dx the threshold should be manually set to 100,000 for N1 and to 10,000 for RP. For other PCR instruments this will be automatically determined.
?. [Sample collection]
Transfer the sample to the laboratory for sample processing. If the time between sample collection and the initial processing steps (aliquoting) is likely to exceed 6 hours, samples can be stored at 2-8°C for up to 7 days, or -80°C for long-term storage, then later thawed on ice for testing.
?. [Sample pretreatment]
Add Proteinase K (see table for volume per sample) to designated 8-strip PCR tubes (200 µL capacity).  ABC1VendorConcentrationVolume2ThermoFisher Scientific MagMAX™ Viral/Pathogen Proteinase K50 mg/mL2.5 µL3New England Biolabs Proteinase K, Molecular Biology Grade20 mg/mL6.25 µL4AmericanBio Proteinase KLyophilized (add 50 mg per 1 mL of nuclease-free water) 2.5 µL
ABC1VendorConcentrationVolume2ThermoFisher Scientific MagMAX™ Viral/Pathogen Proteinase K50 mg/mL2.5 µL3New England Biolabs Proteinase K, Molecular Biology Grade20 mg/mL6.25 µL4AmericanBio Proteinase KLyophilized (add 50 mg per 1 mL of nuclease-free water) 2.5 µL
This work should be completed under BSL-2 conditions, and samples potentially containing SARS-CoV-2 should only be handled in a biosafety cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain SARS-CoV-2.
?. [Sample pretreatment]
Vortex each saliva sample until homogeneous, and immediately transfer 50 µL saliva to each 8-strip PCR tube containing proteinase K. Add 50 µL of nuclease-free water to one 8-strip PCR tube (negative extraction control).
?. [Sample pretreatment]
Close the 8-strip tube lids tightly.
?. [Sample pretreatment]
Place the 8-strip tubes in a rack and vortex for 1 minute at 3000-5000 RPM.
?. [Sample pretreatment]
Briefly spin down the rack/tubes using a plate spinner or 8-strip tube microcentrifuge.
If no plate centrifuge or spinner is available, the plate can be gently tapped to get the samples at the bottom of each well.
?. [Sample pretreatment]
Inactivate the proteinase K by heating samples for 5 minutes at 95°C on a PCR instrument or equivalent thermocycler.
?. [Sample pretreatment]
Briefly spin down the tubes using a plate spinner or 8-strip tube microcentrifuge.
Tubes should only be centrifuged for a few seconds, to spin down condensation in the tubes.
?. [Sample pretreatment]
Store samples at -80°C or proceed immediately to RT-qPCR testing.
?. [Sample pretreatment]
Incubate saliva samples in a thermocycler, water bath, oven or similar at 65°C for 15 minutes, 95°C for 5 minutes OR 95°C for 30 minutes.
If incubating saliva samples at 95°C be sure to use tubes and labels that are stable (i.e. do not burn or melt) at this high temperature.
?. [Sample pretreatment]
Vortex samples then proceed immediately to RT-qPCR testing.
?. [RT-qPCR]
Select the appropriate option below if you are using 96 well plates or 384 well plates.
?. [RT-qPCR]
Add 5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).
Extraction-free saliva samples can be added using a multichannel pipette for high-throughput testing.
?. [RT-qPCR]
Add 5 µL of positive control (Twist synthetic SARS-CoV-2 RNA controls at 100 copies/uL) and no-template control (NTC - water) to designated PCR wells for the controls (1 NTC, and 2 positive controls per plate). Mix by pipetting, taking care to avoid introducing bubbles.
?. [RT-qPCR]
Seal with a transparent plastic qPCR seal. Centrifuge briefly to remove bubbles, if present.
?. [RT-qPCR]
Place the 96-well PCR plate on the PCR cooler and add 15 µL of mastermix to each designated well.
?. [RT-qPCR]
For the Agilent AriaMX: After loading the plate in the thermocycler, place a compression pad securely on the top of the plate and assure the position is aligned with the plate. On the AriaMX screen select “graphical displays” and then adjust or confirm that the Cycle Range for Background Based Threshold is: “8 thru 12”.
?. [RT-qPCR]
On ice, prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting), excluding processed saliva or controls:  Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 11.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free
  water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 11.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free
  water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
?. [RT-qPCR]
Place the 384-well PCR plate on the PCR cooler and add 7.5 µL of mastermix to each designated well.
?. [RT-qPCR]
Add 2.5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).NOTE: processed saliva samples can be added using a multichannel for high-throughput testing
?. [RT-qPCR]
Add 2.5 µL of positive control (e.g. Twist synthetic SARS-CoV-2 RNA controls (100 copies/µL)) and no-template control (NTC - water) to the designated PCR tubes (on ice). Mix by pipetting (avoid bubbles).
?. [RT-qPCR]
Seal with a transparent plastic PCR seal. Briefly centrifuge for a few seconds to remove bubbles, if present.
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
?. [RT-qPCR]
Results will be interpreted according to the table below: For: CFX384 Touch, ABI QuantStudio 5, ABI QuantStudio 6, ABI QuantStudio 7 Pro, ABI QuantStudio 7 Flex, ABI QuantStudio 12K Flex  Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
?. [RT-qPCR]
Prepare 20 uM working stocks of the primers and probes (sequences provided in Materials) by adding 20 µL of 100 uM stock to 80 µL nuclease-free water.
Briefly vortex and centrifuge reagents before use. Probes are photosensitive and should be stored in the dark.
?. [RT-qPCR]
Use the 20 uM working stocks to prepare dualplex primer-probe-water mix containing the following:  ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
A larger volume of primer-probe-water mix can be prepared in advance, aliquoted in LightSafe microcentrifuge tubes, and stored at -20°C.
?. [RT-qPCR]
Select the appropriate option below if you are using 96 well plates or 384 well plates.
?. [RT-qPCR]
Prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting), excluding processed saliva samples or control  Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath  Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -  Primer-probe-water mix (see Step 9.1)    4 µL    4 µL    4 µL  Nuclease-free water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath  Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -  Primer-probe-water mix (see Step 9.1)    4 µL    4 µL    4 µL  Nuclease-free water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
?. [RT-qPCR]
Place the 96-well PCR plate on the PCR cooler and add 15 µL of mastermix to each designated well.
?. [RT-qPCR]
When testing saliva samples immediately in RT-PCR following the pre-treatment heat step (no addition of proteinase K), vortex samples vigorously prior to addition to the PCR plate to ensure samples are homogenous.
After vortexing, samples can be briefly centrifuged for a few seconds prior to opening to draw down any droplets that may have formed.
?. [RT-qPCR]
Add 5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).
Extraction-free saliva samples can be added using a multichannel pipette for high-throughput testing.
?. [RT-qPCR]
Add 5 µL of positive control (Twist synthetic SARS-CoV-2 RNA controls at 100 copies/uL) and no-template control (NTC - water) to designated PCR wells for the controls (1 NTC, and 2 positive controls per plate). Mix by pipetting, taking care to avoid introducing bubbles.
?. [RT-qPCR]
Seal with a transparent plastic qPCR seal. Centrifuge briefly to remove bubbles, if present.
?. [RT-qPCR]
For the Agilent AriaMX: After loading the plate in the thermocycler, place a compression pad securely on the top of the plate and assure the position is aligned with the plate. On the AriaMX screen select “graphical displays” and then adjust or confirm that the Cycle Range for Background Based Threshold is: “8 thru 12”.
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
For the ABI 7500 Fast Dx the threshold should be manually set to 100,000 for N1 and to 10,000 for RP. For other PCR instruments this will be automatically determined.
?. [RT-qPCR]
Report results per the following criteria:  For Bio-Rad CFX96 Touch, ABI 7500 Fast, ABI 7500 Fast Dx and ABI QuantStudio 5  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0  For ABI QuantStudio 6 and ABI Quant Studio 7 Pro  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0  For Agilent AriaMX  Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0  
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<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0
Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0
?. [RT-qPCR]
On ice, prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting), excluding processed saliva or controls:  Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 9.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 9.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
?. [RT-qPCR]
Place the 384-well PCR plate on the PCR cooler and add 7.5 µL of mastermix to each designated well.
?. [RT-qPCR]
Add 2.5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).
Processed saliva samples can be added using a multichannel for high-throughput testing.
?. [RT-qPCR]
Add 2.5 µL of positive control (e.g. Twist synthetic SARS-CoV-2 RNA controls (100 copies/µL)) and no-template control (NTC - water) to the designated PCR tubes (on ice). Mix by pipetting (avoid bubbles).
?. [RT-qPCR]
Seal with a transparent plastic PCR seal. Briefly centrifuge for a few seconds to remove bubbles, if present.
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
?. [RT-qPCR]
Results will be interpreted according to the table below: For: CFX384 Touch, ABI QuantStudio 5, ABI QuantStudio 6, ABI QuantStudio 7 Pro, ABI QuantStudio 7 Flex, ABI QuantStudio 12K Flex  Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
?. [RT-qPCR]
Bring the processed samples and the PCR mastermix plate to a biosafety cabinet.
Most institutions will require samples potentially containing full-length SARS-CoV-2 RNA to be handled in a biosafety cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain full-length SARS-CoV-2 RNA.
?. [RT-qPCR]
Bring the processed samples and the PCR mastermix plate to a biosafety cabinet.
Most institutions will require samples potentially containing full-length SARS-CoV-2 RNA to be handled in a biosafety cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain full-length SARS-CoV-2 RNA.
?. [Sample pretreatment]
Incubate saliva samples in a thermocycler, water bath, oven or similar at 65°C for 15 minutes OR 95°C for 30 minutes.
If incubating saliva samples at 95°C be sure to use tubes and labels that are stable (i.e. do not burn or melt) at this high temperature.
?. [Sample pretreatment]
Add Proteinase K (see table for volume per sample) to designated 8-strip PCR tubes (200 µL capacity).  ABC1VendorConcentrationVolume2ThermoFisher Scientific MagMAX™ Viral/Pathogen Proteinase K50 mg/mL2.5 µL3New England Biolabs Proteinase K, Molecular Biology Grade20 mg/mL6.25 µL4AmericanBio Proteinase KLyophilized (add 50 mg per 1 mL of nuclease-free water) 2.5 µL
ABC1VendorConcentrationVolume2ThermoFisher Scientific MagMAX™ Viral/Pathogen Proteinase K50 mg/mL2.5 µL3New England Biolabs Proteinase K, Molecular Biology Grade20 mg/mL6.25 µL4AmericanBio Proteinase KLyophilized (add 50 mg per 1 mL of nuclease-free water) 2.5 µL
This work should be completed under BSL-2 conditions, and samples potentially containing SARS-CoV-2 should only be handled in a biosafety cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain SARS-CoV-2.
?. [Sample pretreatment]
Vortex each saliva sample until homogeneous, and immediately transfer 50 µL saliva to each 8-strip PCR tube containing proteinase K. Add 50 µL of nuclease-free water to one 8-strip PCR tube (negative extraction control).
?. [Sample pretreatment]
Close the 8-strip tube lids tightly.
?. [Sample pretreatment]
Place the 8-strip tubes in a rack and vortex for 1 minute at 3000-5000 RPM.
?. [Sample pretreatment]
Briefly spin down the rack/tubes using a plate spinner or 8-strip tube microcentrifuge.
If no plate centrifuge or spinner is available, the plate can be gently tapped to get the samples at the bottom of each well.
?. [Sample pretreatment]
Inactivate the proteinase K by heating samples for 5 minutes at 95°C on a PCR instrument or equivalent thermocycler.
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
For the ABI 7500 Fast Dx the threshold should be manually set to 100,000 for N1 and to 10,000 for RP. For other PCR instruments this will be automatically determined.
?. [RT-qPCR]
For the Agilent AriaMX: After loading the plate in the thermocycler, place a compression pad securely on the top of the plate and assure the position is aligned with the plate. On the AriaMX screen select “graphical displays” and then adjust or confirm that the Cycle Range for Background Based Threshold is: “8 thru 12”.
?. [RT-qPCR]
Seal with a transparent plastic qPCR seal. Centrifuge briefly to remove bubbles, if present.
?. [RT-qPCR]
Add 5 µL of positive control (Twist synthetic SARS-CoV-2 RNA controls at 100 copies/uL) and no-template control (NTC - water) to designated PCR wells for the controls (1 NTC, and 2 positive controls per plate). Mix by pipetting, taking care to avoid introducing bubbles.
?. [RT-qPCR]
Add 5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).
Extraction-free saliva samples can be added using a multichannel pipette for high-throughput testing.
?. [RT-qPCR]
Place the 96-well PCR plate on the PCR cooler and add 15 µL of mastermix to each designated well.
?. [RT-qPCR]
Prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting; Table 10), excluding processed saliva samples or control  Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath   Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -  Primer-probe-water mix (see Step 12.1)    4 µL    4 µL    4 µL  Nuclease-fre water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
Component    NEB Luna (2x)    Bio-Rad Reliance    Thermo TaqPath   Master mix    10 µL    5 µL    5 µL   RT    1 µL    -    -  Primer-probe-water mix (see Step 12.1)    4 µL    4 µL    4 µL  Nuclease-fre water    -    6 µL    6 µL  Treated sample or control    5  µL (do not add to master mix)    5 µL (do not add to master mix)    5 µL (do not add to master mix)
?. [RT-qPCR]
Select the appropriate option below if you are using 96 well plates or 384 well plates.
?. [RT-qPCR]
Bring the processed samples and the PCR mastermix plate to a biosafety cabinet.
Most institutions will require samples potentially containing full-length SARS-CoV-2 RNA to be handled in a biosafety cabinet. Please seek guidance from your local biosafety office on specific recommendations for working with samples which could contain full-length SARS-CoV-2 RNA.
?. [RT-qPCR]
Use the 20 uM working stocks to prepare dualplex primer-probe-water mix containing the following:  ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
ABC1ComponentVolume (1 reaction) Volume (100 reactions)22019-nCoV_N1-F (400 nM/reaction)0.4 µL40 µL32019-nCoV_N1-R (400 nM/reaction)0.4 µL40 µL42019-nCoV_N1-P (200 nM/reaction)0.2 µL20 µL5RP-F (150 nM/reaction)0.15 µL15 µL6RP-R (150 nM/reaction)0.15 µL15 µL7RP-P (200 nM/reaction)0.2 µL20 µL8Nuclease-free water2.5 µL250 µL
?. [RT-qPCR]
Prepare 20 uM working stocks of the primers and probes (sequences provided in Materials) by adding 20 µL of 100 uM stock to 80 µL nuclease-free water.
Briefly vortex and centrifuge reagents before use. Probes are photosensitive and should be stored in the dark.
?. [RT-qPCR]
On ice, prepare a master mix from one of the approved vendors containing the following (account for 10% extra lost during pipetting), excluding processed saliva or controls:  Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 12.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
Component    NEB Luna (2x)    NEB Luna 4X    Bio-Rad Reliance    Thermo TaqPath  Master mix  5 µL  2.5 µL  2.5 µL  2.5 µL  RT  0.5 µL    -    -    -  Primer-probe-water mix (see Step 12.1)  2 µL  2 µL  2 µL  2 µL  Nuclease-free water    -  3 µL  3 µL  3 µL  Treated sample or control  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)  2.5 µL (do not add to master mix)
?. [RT-qPCR]
Place the 384-well PCR plate on the PCR cooler and add 7.5 µL of mastermix to each designated well.
?. [RT-qPCR]
Add 2.5 µL of processed saliva sample (including the processed negative extraction control) to each designated well. Mix by pipetting (avoid bubbles).
Processed saliva samples can be added using a multichannel for high-throughput testing.
?. [RT-qPCR]
Add 2.5 µL of positive control (e.g. Twist synthetic SARS-CoV-2 RNA controls (100 copies/µL)) and no-template control (NTC - water) to the designated PCR tubes (on ice). Mix by pipetting (avoid bubbles).
?. [RT-qPCR]
Seal with a transparent plastic PCR seal. Briefly centrifuge for a few seconds to remove bubbles, if present.
?. [RT-qPCR]
Load the plate into the qPCR machine, and run the following thermocycler conditions on standard mode:  ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
ABC1StepTemperatureTime2152°C10 min3295°C 2 min4395°C 10 sec5455°C 30 sec65Read plate (FAM & Cy5 channels)7Repeat steps 3-5 for 44 cycles.
?. [RT-qPCR]
Results will be interpreted according to the table below: For: CFX384 Touch, ABI QuantStudio 5, ABI QuantStudio 6, ABI QuantStudio 7 Pro, ABI QuantStudio 7 Flex, ABI QuantStudio 12K Flex  Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
Result    Ct value N1    Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
?. [Sample pretreatment]
Store samples at -80°C or proceed immediately to RT-qPCR testing.
?. [RT-qPCR]
Report results per the following criteria:  For Bio-Rad CFX96 Touch, ABI 7500 Fast, ABI 7500 Fast Dx and ABI QuantStudio 5  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0  For ABI QuantStudio 6 and ABI Quant Studio 7 Pro  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0  For Agilent AriaMX  Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0  
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<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0
Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0
?. [RT-qPCR]
Report results per the following criteria:  For Bio-Rad CFX96 Touch, ABI 7500 Fast, ABI 7500 Fast Dx and ABI QuantStudio 5  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0  For ABI QuantStudio 6 and ABI Quant Studio 7 Pro  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0  For Agilent AriaMX  Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0
<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0
Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0
?. [RT-qPCR]
Report results per the following criteria:  For Bio-Rad CFX96 Touch, ABI 7500 Fast, ABI 7500 Fast Dx and ABI QuantStudio 5  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0  For ABI QuantStudio 6 and ABI Quant Studio 7 Pro  <b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0  For Agilent AriaMX  Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0  
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<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;40.0    Any value    Negative    ≥40.0    &lt;35.0    *Invalid    ≥40.0    ≥35.0
<b style="mso-bidi-font-weight:
  normal">Result    <b style="mso-bidi-font-weight:
  normal">Ct value N1    <b style="mso-bidi-font-weight:
  normal">Ct value RP    Positive    &lt;37.0    Any value    Negative    ≥37.0    &lt;35.0    *Invalid    ≥37.0    ≥35.0
Result    Cq*** value N1    Ct value RP    Positive    &lt;34.0    Any Value    Negative    ≥36.0    &lt;30.0    **Inconclusive    ≥34.0 - &lt;36.0    &lt;30.0    *Invalid    ≥34.0    ≥30.0
?. [RT-qPCR]
When testing saliva samples immediately in RT-PCR following the pre-treatment heat step (no addition of proteinase K), vortex samples vigorously prior to addition to the PCR plate to ensure samples are homogenous.
After vortexing, samples can be briefly centrifuged for a few seconds prior to opening to draw down any droplets that may have formed.