TITLE: Rosbash/Janelia StickLAMP Protocol
AUTHORS: Albert Yu, Tim Brown, Jasmine Quynh Le, Kristina Galatsis,  Michael Rosbash

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for the detection of SARS-CoV-2 from saliva samples featuring a rapid purification step and a high-contrast colorimetric readout. Saliva is first inactivated using a 100x inactivation reagent consisting of 2.5M TCEP, 100 mM EDTA, 1.2N NaOH solution diluted to approximately 1x final concentration and heated to 95C for 5 minutes. RNA is rapidly purified and concentrated with magnetic beads in a PEG/NaCl-based buffer using a 3D-printed magnetic stick that enables selective separation of beads without carryover of saliva contaminants. Beads are eluted directly into an RT-LAMP reaction mix, which uses a novel high contrast dye that turns from purple to clear when acidified by nucleic acid amplification products that enables unambiguous identification of successful amplification. This protocol is sensitive down to 1 copy/µl of SARS-CoV-2 in 300 µl of saliva. This degree of sensitivity enables faithful detection of SARS-CoV-2 even in pooled samples.</div></div>

[STEPS]
?. [Saliva Collection]
Instruct patient to avoid food, drink, toothbrushing, and nasal sprays for a minimum of   prior to sample collection
?. [Saliva Collection]
Begin pooling saliva in your mouth. Saliva production can be stimulated by thinking about food, or about the saliva collection itself.
?. [Saliva Collection]
Gently expel saliva into the funnel, tapping to collect in the tube, until amount of saliva is approximately flush with the base of the funnel
[Approximately]
?. [Inactivation]
Add inactivation reagent to approximately 1x final concentration. Reaction is tolerant of between 0.7x to 2x final concentration.
[Approximately]
?. [Inactivation]
Invert 40 times to mix
?. [Inactivation]
Heat tube to approximately   for  . Viral RNA release is similar between 93-98C. Use tube clip to prevent popping.
95 °C
?. [Inactivation]
Remove tube from heat and let rest at   for at least   OR   for at least .
25 Room temperature
on ice
?. [Quick Purification and LAMP Reaction]
While tube is resting, aliquot   SARS-CoV-2 mastermix and   Actin mastermix to separate wells of PCR strip tube, 96-well plate, or 1.5ml tube per sample . Per run, prepare two additional   SARS-CoV-2 mastermixes for positive and negative controls.
25 µl
25 µl
on ice
25 µl
?. [Quick Purification and LAMP Reaction]
Add approximately 0.7x volumes of bead mix -  . Sample is tolerant of between 0.7x-1.2x volumes of bead mix. Pipette up and down to mix.
[Approximately]
?. [Quick Purification and LAMP Reaction]
Let stand at   for
0 Room temperature
?. [Quick Purification and LAMP Reaction]
Cap magnetic stick with a clean tip and dip in bead/sample mix for  , dipping up and down 5 times every  . Meanwhile, prepare  130mM NaCl in a separate 1.5ml or 2ml tube.
500 µl
?. [Quick Purification and LAMP Reaction]
Remove magnetic stick from sample and swirl in clean 130mM NaCl solution for  . Discard NaCl solution.
?. [Quick Purification and LAMP Reaction]
Remove magnetic stick from wash sample and place in SARS-CoV-2 mix for
?. [Quick Purification and LAMP Reaction]
Remove magnetic stick from SARS-CoV-2 mix and place in Actin mix for
?. [Quick Purification and LAMP Reaction]
Add   water to additional SARS-CoV-2 Mix (negative control) and   synthetic Twist SARS-CoV-2 positive RNA control to additional SARS-CoV-2 Mix, prepared in Step 8.
5 µl
5 µl
?. [Quick Purification and LAMP Reaction]
Cap tubes and place on   heating apparatus for  . If using a thermal cycler, run with the following program:65C for 40 minutes4C indefinitely
65 °C
?. [Quick Purification and LAMP Reaction]
Remove tubes from heating apparatus and examine color change.
?. [Quick Purification and LAMP Reaction]
If a positive sample is found when pooling, re-test pooled samples individually.