TITLE: CoVan RT-LAMP protocol
AUTHORS: David O'Connor, Shelby O'Connor, Amelia Haj, Mitchell Ramuta, Christina Newman, Dawn Dudley

[STEPS]
?. [Saliva collection area]
Prepare saliva collection tube by placing a p1000 pipet tip in a 1.5 ml tube
?. [Saliva collection area]
Obtain ~300 µl of saliva – have the individual spit into a tube using a P1000 pipet tip to funnel saliva into the tube
Infection control: Have individual throw the p1000 pipet tip away in a biohazard bag, close the cap of the 1.5ml tube, and wipe down the outside of the tube with 10% bleach
?. [Saliva collection area]
Place the saliva sample in a cooler with ice packs and transport the saliva to the sample preparation table
?. [Sample preparation area]
Heat saliva sample at 65ºC for 30 minutes, while shaking gently at 300 rpm, to inactivate virus
Infection control: Wipe down the outside of the tube again with 10% bleach.
This is a good time to start preparing the RT-LAMP master mix (step 9)
?. [Sample preparation area]
Heat and shake sample at 98ºC for 3 minutes to liberate RNA and help inactivate some of the enzymes in saliva
?. [Sample preparation area]
Spin down tubes in microcentrifuge for 2 minutes to pellet cell debris in the saliva
Infection control: Wipe down the microcentrifuge with 10% bleach and 70% ethanol after spin; wipe down tubes with 10% bleach
?. [Sample preparation area]
Add 50 µl of saliva to a 1.5 ml Sarstedt tube with 50 µl of 1x PBS to buffer against variability in pH and make the sample easier to handle
Infection control: Wipe down the tubes with 10% bleach
?. [Sample preparation area]
Transport the saliva + PBS tubes to the RT-LAMP testing area
Infection control: Wipe down the tubes with 10% bleach
?. [RT-LAMP area]
Prepare RT-LAMP master mix:  ABC1ReagentMaster mix (1 reaction)Your reaction volumes:2WarmStart 2X MM10 µl3LAMP primer mix (10X)2 µl4dH2O5 µl5Sample3 µl6Total Volume20 µl
ABC1ReagentMaster mix (1 reaction)Your reaction volumes:2WarmStart 2X MM10 µl3LAMP primer mix (10X)2 µl4dH2O5 µl5Sample3 µl6Total Volume20 µl
?. [RT-LAMP area]
Aliquot 17 µl of RT-LAMP master mix into a new set of PCR strip tubes – two for each sample, plus two each for positive and negative controls
?. [RT-LAMP area]
Add 3 µl of saliva/PBS mix to 17 µl RT-LAMP master mix in duplicate – two tests per individual
Infection control: Wipe down the tubes with 10% bleach
?. [RT-LAMP area]
For a positive control, add 3 µl of a 1e6 copies/ml UV-inactivated SARS-CoV-2 stock to two 17 µl RT-LAMP master mix tubes. (More information on how to prepare a positive control stock here.) For a negative control, add 3 µl of water to two 17 µl RT-LAMP master mix tubes.
?. [RT-LAMP area]
Heat and shake sample + master mix at 65ºC for 30 minutes
?. [RT-LAMP area]
Observe RT-LAMP reaction result for each individual. All four controls (two positive and two negative) must be correct in order to consider results valid. If any of the controls don't work, repeat the entire run. Both sample tubes are PINK: negative resultBoth sample tubes are YELLOW: positive resultOne sample tube is PINK and one sample tube is YELLOW: positive, but retest –Re-run sample 4x using the same PBS/saliva mixOptional PCR confirmation of results