TITLE: One Hour Covid Test Protocol
AUTHORS: Sarah Ware, Ellen Jorgensen, Chris Monaco

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a fast (one hour), very simple LAMP-based Covid-19 diagnostic test using readily-available components that is appropriate for point-of-care use and also can be easilty scaled up on a pipetting robot such as an Opentrons OT2 because the protocol requires no cengtrifugation.</div><div class = "text-block">It leverages a commercially-avaialble swab kit to collect a sample from the mouth of the patient in a safe manner. Viruses are inactivated by the buffer in the swab kit so risk of exposure is minimal. The sample can be safely handled once the swab is placed back in the collection tube. The collection kit has an EUA from FDA.</div><div class = "text-block">A commercially-available reagent kit called prepIT.Q2A is used to prepare an aliquot of the sample for LAMP analysis. The two kit reagents are added to 100uL of the sample, a precipitate forms and falls to the tube bottom with no centrifugation necessary, and the RNA is pipetted off the top of the liquid in the tube.</div><div class = "text-block">The RNA solution is diluted 1:10 with water and analyzed by adding 1uL to a preloaded PCR tube with primers and the NEB WarmStart Colorimetric LAMP reagent. Incubation at 68C for 30min produces a color change visible to the eye for a simple yes/no readout.</div><div class = "text-block">The whole process takes an hour from start to finish.</div></div>

[STEPS]
?. [OPTIONAL pre-preparation of Master Mix]
LAMP Master Mix preparation (OPTIONAL, supplied premixed in our kit)NOTE: Kits will have LAMP Master Mix already prepared with guanidine and primers in it in PCR tubes. We are leveraging the work of scientists at New England Biolabs and include the preparation because of our shared commitment to open science. This is an open source protocol and anyone can buy the materials and make it themselves. Please wear proper lab gloves and safety glasses when preparing this. The primers, water and NEB WarmStart Colormetric Master Mix do not contain any harsh chemicals but take appropriate care in making the 1M guanidine HCl, especially if you need to adjust the pH with sodium hydroxide. Here is what you need- these things are common labware and can be purchased from a variety of vendors without affecting the quality of the Master Mix, so equivalents can be used:
?. Purchase LAMP primer sets from IDT or other oligo synthesis company. You will need three sets of primers. Two for detecting Covid-19 and one as a control to show that you have successfully extracted RNA from human samples. The primers can be changed to reflect the latest consensus of scientific opinion on the best primers to use. In this iteration we will use actinR for control primers and the N2/E1 set of primers developed by New England Biolabs. LAMP primer sets have six primers each, and they are designated BIP, FIP, F3, B3, LoopF (LF) and LoopB (LB).The primer sequences (read 5prime to 3prime) are as follows:For N2 Primer mix:  N2-F3       ACCAGGAACTAATCAGACAAG  N2-B3      GACTTGATCTTTGAAATTTGGATCT  N2-FIP     TTCCGAAGAACGCTGAAGCGGAACTGATTACAAACATTGGCC  N2-BIP     CGCATTGGCATGGAAGTCACAATTTGATGGCACCTGTGTA  N2-LF      GGGGGCAAATTGTGCAATTTG  N2-LB      CTTCGGGAACGTGGTTGACC  For E1 Primer Mix:  E1-F3      TGAGTACGAACTTATGTACTCAT  E1-B3      TTCAGATTTTTAACACGAGAGT  E1-FIP     ACCACGAAAGCAAGAAAAAGAAGTTCGTTTCGGAAGAGACAG  E1-BIP    TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT  E1-LB      GCGCTTCGATTGTGTGCGT  E1-LF      CGCTATTAACTATTAACGFor actinR Primer Mix:ACTB-F3    AGTACCCCATCGAGCACG ACTB-B3    AGCCTGGATAGCAACGTACA ACTB-FIP   GAGCCACACGCAGCTCATTGTATCACCAACTGGGACGACA ACTB-BIP  CTGAACCCCAAGGCCAACCGGCTGGGGTGTTGAAGGTC ACTB-LF    TGTGGTGCCAGATTTTCTCCA ACTB-LB    CGAGAAGATGACCCAGATCATGT
?. Resuspend Primers.When primers arrive they will be in plastic screw-cap vials in a dry form. Set the vials in the tube rack and for each vial use the P-200 or P-1000 pipettor and appropriate tips add nuclease-free water to achieve a final primer concentration of   . A rule of thumb is to add ten times the number of microliters as there are nanomoles of primer in the vial. For example, if the vial label says it contains 20.3 nanomoles of primer DNA, add 203 microliters of water. Store frozen at  until ready to use them.
-20 °C
?. Make 10x Primer Mix.You will need one 1.5mL microfuge tube for each of the 3 sets of primers. Make separate 10x mixes for each by placing the tubes in the tube rack and using the P-10, P-20 and P-200 pipettors and appropriate tips to Make separate 10X Primer Mixes for each primer set.  To make   you must addNote that the amount can be scaled up by multiplying all component volumes by the same value (e.g. ten times each component will give you one milliliter final volume of 10x Primer Mix). Store frozen at  until ready to use.
[10x Primer Mix]
[BIP]
[FIP]
[F3]
[B3]
[LoopF]
[LoopB]
[nuclease free water]
-20 °C
?. Prepare 1M Guanidine HClTo make  , use the weigh boat and analytical balance to weigh out  . Place in a 15mL conical tube in rack. Using the 10mL serological pipette and pipette controller (NEVER pipette by mouth) add   to the tube, cap and invert several times to dissolve the guanidine. Adjust pH to around 7 using sodium hydroxide and test strips if needed.
[Guanidine HCL]
[Guanidine HCl]
[nuclease free water]
?. Prepare and Aliquot the LAMP Master MixPlace a 1.5mL tube in the 1.5mL tube rack and using the pipettors add the following for 1mL Master Mix with Covid Primers: 1Place PCR tubes in rack. Using the p-20 pipettor and tips, dispense the Master Mix into 0.2mL PCR tubes in the amount of Store frozen at  until use.
[NEB 2x Colorimetric LAMP Master Mix]
[N2 Primer Mix]
[E1 primer mix]
[1M Guanidine HCl]
[nuclease free water]
[per tube]
-20 °C
?. [Test Protocol: Sample Collection]
Using the DNA Genotek OR-100 (aka ORE-100) swab kit for RNA collectionIn developing the simplest kit, we decided to take advantage of the fact that DNA Genotek has already gotten an EUA apporval for these kits, and they have already tested and confirmed that viruses are inactivated and the RNA preserved for analysis when the swab is submerged in the kit buffer. Thus they are sfe and effective.Additionally, we chose swabs rather than liquid saliva collection because in talking with nursing homes many of their residents cannot produce much saliva. Furthermore physicians told us that it is necessary to test patients who are on ventilators and unconcious and in these cases saliva collection is impossible but swabbing is doable.
?. [Test Protocol: Sample Collection]
Open the individually-wrapped swab kit, taking care not to touch the swab itself. Note that when swabbing someone other than oneself, appropriate PPE such as gloves, face shields, medical coveralls and N95 masks may be necessary for safety.
?. [Test Protocol: Sample Collection]
Swab towards the back of the throat for about ten seconds. Note that the swab remains attached to the top of the tube during this time.
?. [Test Protocol: Sample Collection]
Carefully unscrew the cap and reverse it, sealing the swab inside. Do not touch the swab during this procedure. Make sure the cap is securely tightened.
?. [Test Protocol: Sample Collection]
Shake the tube vigorously for five seconds.
?. [Test Protocol: Sample Collection]
Store at  until ready to assay.
0 Room temperature
?. [Test Protocol: Sample Assay]
Assaying the sampleIn developing this assay we took into account multiple considerations including speed, ease-of-use and minimizing expensive lab equipment. We tried literally a dozen different methods that addressed how to go from a human sample into the pH-dependent colorimetic LAMP assay that can be read with no special equipment. In the end it was a conversation between us and our DNA Genotek rep that sparked the idea of trying a reagent that they sell. It is sold and normally used for DNA extraction and nobody had ever tried it for viral RNA. We obtained samples of it and it worked beautifully. The best part is it is not only fast but requires no centrifugation. This opens up great possibilities for scale-up becuase the whole procedure can be done on a lab robot with no need to remove tubes and spin them.You will need:Our kit, which will include:2 PCR tubes prefilled with LAMP MAster Mix + guanidine and primers, one with Covid primers N2/E1 and one with actinR control primersTube containing  nuclease-free molecular biology grade waterand A way to heat the tubes to   for This could be anything that achieves this- a heat block, a water bath etc. We used a cheap sous vide cooker set we got (sold on Amazon, Target and BestBuy). Buy the heater and lid separate and set them on top of any thing of appropriate size that holds water and can take 68C temperatures. on top of an ordinary kitchen pot, total cost less than $150.Other stuff you will need will be:
270 µl
68 °C
?. [Test Protocol: Sample Assay]
Turn on sous vide heater and set to  .Place a 1.5mL microfuge tube in the tube rack. Using the P-200 pipettor and filter tips, transfer 100uL of the liquid  from the OR-100 kit used for the sample into the microfuge tube.
68 °C
?. [Test Protocol: Sample Assay]
Using the P-20 pipettor and tips, add  prepIT.Q2A Solution AG
10 µl
?. [Test Protocol: Sample Assay]
Using the P-20 pipettor and tips, add   prepIT.Q2A Solution ST
20 µl
?. [Test Protocol: Sample Assay]
Pipette up and down 12 times to mix.
?. [Test Protocol: Sample Assay]
Incubate tube  for A precipitate will form and settle to the tube bottom, leaving a clear aqueous layer on top.
0 Room temperature
?. [Test Protocol: Sample Assay]
Using the P-200 pipettor and tips, transfer   from the top of the aqueous phase to the tube with 270 µl nuclease-free water supplied in the kit.This is your working dilution.
30 µl
?. [Test Protocol: Sample Assay]
Thaw the two tubes from the kit containing LAMP Master Mix, one with N2/E1 primers and one with the control  actinR primers. Using the P10 pipettor and tips, add  of the working dilution from step 3.6 to each PCR tube.
1 µl
?. [Test Protocol: Sample Assay]
Place the PCR tubes in a floating rack in the  sous vide waterbath and incubate for
68 °C
?. [Assay Readout]
The tubes can be read by eye. Remove the tubes from the waterbath and observe the color. Yellow indicates that the target sequence is present.If the actinR control tube is not yellow, discard the test result. It means that ther was no human RNA collected and there was probably something wrong with the sample collection or preparation.A true negative result is the N2/E1 PCR tube remaining pink while the actinR control turns yellow.A true positve result is when both tubes turn yellow.This procedure can be automated by the use of a 96-well plate and a plate reader in place of individual PCR tubes. In that case it may be necessary to provide the kit with Master Mix made using NEB WarmStart®Colorimetric LAMP 2X Master Mix with UDG to prevent cross-contamination in plates.