.3 viral copies per reaction. 
T--TTTTTAG TAG
A--TTGTTAA CGT Supplement S1. Vacuum manifold (optional) 3.
Materials required for viral nucleic acid extraction as recommended by the extraction kit protocol 4.
96-100% ACS grade ethanol 5.
Warmstart Colorimetric LAMP 2X Master Mix (NEB, M1800S, 100 reactions) 6.
Six primers (F3, B3, FIP, BIP, LoopF, LoopB) 7.
Microcentrifuge (adjustable, up to 8000 × g) 8.
Heat block with lid/heated lid or thermal cycler (for 60 o C incubation) 9.
Thermometers (for confirming temperature of heat block during incubation) 10.
Adjustable pipettes (10, 20, 100, 200, 1000 μL) 11.
Sterile, RNase-free pipette tips with aerosol barrier 12.
PCR-grade water 13.
75% ethanol or 2% Virkon for disinfection
1. All procedures should be performed in Biosafety Level 2 (BSL-2) laboratory, or in a separate ventilated compartment of a mobile/temporary diagnostic unit.
All procedures involving non-inactivated clinical samples should be performed inside a validated class II biosafety cabinet or a sample processing chamber with UV lamp in a mobile/temporary diagnostic unit if it is available.
Laboratory gown and gloves shall be worn at all procedures. Sleeves, disposable gloves and surgical mask shall be worn when non-inactivated specimens are being handled, in a BSL-2 laboratory with class II biosafety cabinet setting.
If a BSL-2 laboratory or class II biosafety cabinet is not available, for example in a mobile or temporary diagnostic unit, personal protection equipment (PPE) including N95 masks, safety googles, face shields, disposable lab gowns, gloves and hair covers should be worn before inactivation of the virus in the samples.
After completing all procedures, discard the PPE carefully inside the biosafety cabinet or a UV chamber (if it is available) and package and tie/seal them with plastic bags. Wash hands with soap/hand rub with 70-75% ethanol immediately for at least 30 seconds.
Respiratory specimens: Nasopharyngeal swabs/aspirates, sputa/deep throat saliva, throat swabs in transport medium or viral inactivation collection tubes. Specimens should be kept at 2-4°C or extract within four hours.
(Outside biosafety cabinet or sample processing UV chamber) 1.
Prepare the lysis buffer as instructed by the manufacturer's protocol. (Add 5.6 L of carrier RNA in each 0.56 mL of AVL buffer, mix by inverting the tube 5-10 times. DO NOT VORTEX.) 2.
Aliquot appropriate amount of lysis buffer (560 L of AVL buffer) into each screw cap tubes.
Transfer the screw cap tube (with lysis buffer) to biosafety cabinet/sample processing UV chamber.
(Inside biosafety cabinet/UV chamber) 4. Aliquot the samples into the screw cap tubes containing the lysis buffer (140 L if using QIAamp Viral RNA Mini Kit).
Mix by pipetting up and down 5 times and inverting up and down 5 times. Incubate the mixture at room temperature for 2 minutes.
Disinfect the inactivated sample tubes with 2% Virkon/75% ethanol before taking out from biosafety cabinet/UV chamber. 7.
Store remaining specimen bottles in a large plastic double bag, labelled with date of requested for temporary storage. 8.
Disinfect the biosafety cabinet/UV chamber using 2% Virkon or 75% ethanol after use.
(Outside biosafety cabinet/UV chamber) 9. Spin down the lysate. 10.
Add 560 L of 96-100% ACS grade ethanol to the sample. Mix by vortexing/inverting and spinning down. 11.
Add 630 L of the mixture to column, spin at 8000 × g for 30 s (or using a vacuum manifold to pull down the samples). Repeat this step until all mixture has been loaded into column. 12.
Discard the flow-through. Add 500 L of AW1, spin at 6000 × g for 30 s (or using a vacuum manifold to pull down the washing solutions). 13.
Discard the flow-through. Add 500 L of AW2, spin at full speed for 30 s (or using a vacuum manifold to pull down the washing solutions). 14.
Place the column into a new 2 mL collection tube, dry spin at full speed for 1 min (or using a vacuum manifold to pull down the washing solutions) to remove residual AW2. 15.
Place column to a 1.5 mL tube. Add 60 l of AVE buffer. Incubate at room temperature for 1 min.
Spin at 8000 × g for 1 min to collect the RNA.
The extracted RNA tubes should be disinfected by 2% Virkon/75% ethanol before passing to the LAMP reaction room/compartment for safety concern. 17.
The extracted RNA will be passed in an air-sealed UV pass box or in a clean secondary container to the LAMP reaction room/compartment. 
For each 1.25 mL of Warmstart colorimetric LAMP 2× Mastermix, 750 µ L of primer mix will be added to it. 20.
Pre-aliquot each 20 µ L of the reaction mix to a 0.5 mL tube. (store under −20°C for long-term storage or under 4°C for use within one day) 21.
Label reaction tubes according to samples list.
Label positive and negative control tubes.
Add 5 l of RNA or controls to each LAMP reaction tube for final reaction volume of 25 l.
Spin down the tubes if needed. 23.
Place the colorimetric RT-LAMP reaction tubes in a 60°C heat block with lid/heated lid or thermal cycler for a maximum of 90 min. Thermometers should be used to confirm the temperature. Condensation can be minimized if heat block with heated lid or thermal cycler is used. If a heat block without lid is used, wrap the heat block with aluminum foil and utilize good thermal insulation materials such as polyurethane/polystyrene foam, cellulose, fiberglass and mineral wool as temporary lid. 24.
Quick spin before result inspection to avoid condensation of solutions on top of tubes. 25.
Inspect result only at 30 min, 60 min and 90 min to avoid temperature disturbance. (To avoid amplicon contamination, DO NOT open the LAMP reaction tubes after addition of RNA.)
If the color in the reaction is changed from pink to yellowish-orange or yellow at either 60 min or 90 min, it is regarded as a positive result, i.e. SARS-CoV-2 RNA is detected (see photo below). At 60 min, if the color is changed from pink to orange or remained unchanged, it is