TITLE: High-throughput Sars-CoV-2  detection from self-collected saline gargle samples
AUTHORS: Noel Rouven Stierlin, Laura   Laks, Regina   Reimann, Miodrag  Savic, Fabian  Rudolf, Adriano  Aguzzi, Karl Frontzek

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The corona virus disease 19 (COVID19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide health crisis. A rigid test, trace and isolate strategy is obstructed by a global shortage in lab supplies such as RNA extraction kits, RT-PCR reagents or other consumables. We opted to develop a cheap and easily scalable molecular Sars-CoV-2 test.</div><div class = "text-block">Nasopharanygeal and/or throat swabs and subsequent quantitative RT-PCR (RT-qPCR) are the current gold standard for detection of SARS-CoV-2. Swabs need to be taken, however, by skilled personell and exposure to infected individuals poses a serious health risk to health care professionals. </div><div class = "text-block">Here, we describe a protocol for high-throughput Sars-CoV-2 detection from self-collected saline gargle samples, a rapid (collection to result < 3 hours), scalable (up to several 1'000 samples per day) and sensitive (10 copies Sars-CoV-2 per reaction) molecular test that based on self-collected saliva. Unstimulated saliva after wake-up or fasting for 1 hour is collected by gargling with common saline (0.9% NaCl) solution. Saliva is stored in plastic buckets without additives and is stable even after 12 hours at 4°C plus at least one freeze-thaw cycle. </div><div class = "text-block">When compared to the best available method for Sars-CoV-2 detection, our protocol reaches a positive percent agreement of 95 % and a negative percent agreement of 98.9 % at a cost of ca. 5 $ per test.  Rapidly scalable, molecular detection of Sars-CoV-2 from saline/saliva mixtures using alternative reagents could help reduce disease burden when implemented in a test, trace and isolate regimen. </div></div>

[STEPS]
?. [Prepare reagents and instruments for nucleic acid extraction]
Prepare GdnHCl  / Chelex mix. Note: GdnHCl helps to stablize the RNA at 65°C during Chelex incubation. Prepare per sample plus 15% overage:   GdnHCl  +  Chelex 30%.
250 µl
900 µl
CHELEX beads settle quickly and need to be resuspended directly prior to pipetting
?. [Nucleic acid extraction ** IN BSL2 LAMINAR FLOW HOOD **]
Add saliva to Chelex / GdnHCl mix.Using a multichannel pipette, add  of saliva-saline solution to  CHELEX / GdnHCl.Mix thouroughly.
450 µl
1150 µl
?. [Prepare reagents and instruments for nucleic acid extraction]
Prepare nucleic acid extraction plate:  - 96 deep well plate.Pre-fill wells with  GdnHCl / CHELEX mix.Put on ice  .
2 mL
2.5 mL
1150 µl
on ice
?. [Prepare reagents and instruments for nucleic acid extraction]
Prepare SpeedBeads in SPRI RNA bead binding buffer.For of SpeedBeads, add  of SPRI RNA bead binding buffer. Bead suspension can be stored at  for 4 weeks.Prepare per sample + 15% overage SpeedBead-MixPut
SpeedBeads need to be vortexed thouroughly before pipetting to ensure a good suspension
100 µl
4.9 mL
4 °C
480 µl
on ice
?. [Prepare reagents and instruments for nucleic acid extraction]
Set plate thermoshaker at  .
65 °C
?. [Nucleic acid extraction ** IN BSL2 LAMINAR FLOW HOOD **]
Seal 96 deep well plate tightly with aluminium plate seal or sealing mat.
Ensure that no cross-contamination can occur during shaking
?. [Nucleic acid extraction ** IN BSL2 LAMINAR FLOW HOOD **]
Incubate samples for  at  under constant shaking at - and put .
65 °C
33
33
on ice
?. [Magnetic bead clean up]
Briefly spin down 96 deep well plate at  for  to pellet CHELEX resin.
Centrifuge: 200 34
?. [Magnetic bead clean up]
Transfer  of supernatant from CHELEX / GdnHCl incubation to SpeedBead-Mix, resuspend thouroughly.
600 µl
?. [Magnetic bead clean up]
Prepare a fresh -96 deep well plate with of SpeedBead-Mix per well.
1.1 mL
1.5 mL
480 µl
?. [Magnetic bead clean up]
Seal plate, incubate for  at .During incubation, prepare fresh 80% EtOH per sample in nuclease-free water.
65 Room temperature
1 mL
?. [Magnetic bead clean up]
Put deep well plate on magnetic rack, wait for  or until all beads have pelleted.
?. [Magnetic bead clean up]
Remove supernatant, add   of 80% EtOH to beads, briefly shake plate and repeat this step once.
500 µl
Shake plate gently to avoid cross-contamination.
?. [Magnetic bead clean up]
Remove residual EtOH, dry plate for  or until beads no longer look "shiny".
Do not let beads overdry and crack
?. [Magnetic bead clean up]
Remove from magnet, add  Tris   per well.Incubate for  at .
100 µl
0 Room temperature
Resuspend well
?. [Magnetic bead clean up]
Pellet on magnet for .Collect up to 75 µl of supernatant in clean tubes or plates and immediately proceed with RT-qPCR. Alternatively, samples can be stored at -80°C for further use.Technical note: We observed that it may help leave some elution buffer in the plate to avoid resuspension of beads.
?. [Prepare reagents and instruments for nucleic acid extraction]
Prepare bead binding bufferFor a 1x working solution mix following chemicals:ItemConc. (stock)VolumeConc. (final)Sodium Chloride5 Molarity (M)25 mL2.5 Molarity (M)Nuclease-free Water 4.672 mLTrisodium citrate1 Molarity (M)50 µL1 Milimolar (mM)Tween 2020% w/v250 µL0.05% volumePEG 800050% w/v20 mL20% volumeHCl1 Molarity (M)28 µL0.56 Milimolar (mM)Titrate the Buffer to  with HCl.Approximately  of 37% HCl for a  Volume.
ItemConc. (stock)VolumeConc. (final)Sodium Chloride5 Molarity (M)25 mL2.5 Molarity (M)Nuclease-free Water 4.672 mLTrisodium citrate1 Molarity (M)50 µL1 Milimolar (mM)Tween 2020% w/v250 µL0.05% volumePEG 800050% w/v20 mL20% volumeHCl1 Molarity (M)28 µL0.56 Milimolar (mM)
35 µl
100 mL
Link: https://openwetware.org/wiki/SPRI_bead_binding_buffer
?. [Prepare reagents and instruments for nucleic acid extraction]
Prepare a 30% w/v Chelex stock solution:For 16 Samples add  Chelex to a 15 ml falcon tube. Fill to  with nuclease-free, deionized water. Shake the tube well until no clumps are visible. Chelex stock solution is stable at 4°C for up to 4 weeks.
4.5 g
15 mL
?. [RT-PCR]
For the PCR step, following primers are used:NameSequence:5'->3'Stock solution [µM]
Stock solution for primer master mix [µl]
Final reaction conc.
[nM]
CDC N1 fwdGAC CCC AAA ATC AGC GAA AT1004400CDC N1 revTCT GGT TAC TGC CAG TTG AAT CTG1004400CDC N1 FAMACC CCG CAT TAC GTT TGG TGG ACC1002200Charité E-Sarbeco fwdACA GGT ACG TTA ATA GTT AAT AGC GT1004400Charité E-Sarbeco revATA TTG CAG CAG TAC GCA CAC A1004400Charité E-Sarbeco HEXACA CTA GCC ATC CTT ACT GCG CTT CG1002200

CDC RNase P fwdAGA TTT GGA CCT GCG AGC1004400CDC RNase P revGAG CGG CTG TCT CCA CAA GT1004400CDC RNase P Cy5TTC TGA CCT GAA GGC TCT GCG CG1002200
NameSequence:5'->3'Stock solution [µM]
Stock solution for primer master mix [µl]
Final reaction conc.
[nM]
CDC N1 fwdGAC CCC AAA ATC AGC GAA AT1004400CDC N1 revTCT GGT TAC TGC CAG TTG AAT CTG1004400CDC N1 FAMACC CCG CAT TAC GTT TGG TGG ACC1002200Charité E-Sarbeco fwdACA GGT ACG TTA ATA GTT AAT AGC GT1004400Charité E-Sarbeco revATA TTG CAG CAG TAC GCA CAC A1004400Charité E-Sarbeco HEXACA CTA GCC ATC CTT ACT GCG CTT CG1002200

CDC RNase P fwdAGA TTT GGA CCT GCG AGC1004400CDC RNase P revGAG CGG CTG TCT CCA CAA GT1004400CDC RNase P Cy5TTC TGA CCT GAA GGC TCT GCG CG1002200
?. [RT-PCR]
Following table is for one PCR reaction:TaqPath™ 1-Step Multiplex Master Mix (No ROX)2.5 µl
 Primer Mix (step 17)1 µl
 Nuclease-free water1.5 µl
 Sample5 µl
 Total10 µl
 Prepare .
TaqPath™ 1-Step Multiplex Master Mix (No ROX)2.5 µl
 Primer Mix (step 17)1 µl
 Nuclease-free water1.5 µl
 Sample5 µl
 Total10 µl
on ice
The primers are light sensitive. Avoid long exposure to light. Cover up with aluminum foil. Use RNAse + nucleic acid free tubes.
?. [RT-PCR]
Load onto a RT-PCR plate - we usually run all samples and controls in duplicates.Note: we used 500 copies of TWIST synthetic RNA as positive control and nuclease-free water as negative control.Spin down quickly before loading the plate into the RT-PCR machine.
?. [RT-PCR]
PCR Program used will take :UNC incubation and Hold Stage:PCR Stage:Repeat for 45 cycles.
25 °C
53 °C
95 °C
95 °C
55 °C
?. [RT-PCR]
Interpretation of resultsE Ct 
(both replicates)N1 Ct 
(both replicates)RNase P Ct 
(both replicates)Interpretation≤ 40≤ 40positiveanyany≥ 35invalid> 40> 40negative
E Ct 
(both replicates)N1 Ct 
(both replicates)RNase P Ct 
(both replicates)Interpretation≤ 40≤ 40positiveanyany≥ 35invalid> 40> 40negative