Sep 08, 2020
Version 1
One Hour Covid Test Protocol V.1
- Sarah Ware1,
- Ellen D Jorgensen2,
- Chris Monaco3
- 1[Bioblaze];
- 2Aanika Biosciences;
- 3Centers for Disease Control
- XPRIZE Rapid Covid Testing
- Open Science
Protocol Citation: Sarah Ware, Ellen D Jorgensen, Chris Monaco 2020. One Hour Covid Test Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bktakwie
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41538
Keywords: Covid-19, diagnostic, fast, isothermal amplification, LAMP,
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
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.
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.
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.
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.
The whole process takes an hour from start to finish.
Guidelines
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Materials
MATERIALS
primers
Water, nuclease-freeThermo FisherCatalog #R0581
Sodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
Guanidine hydrochlorideSigma AldrichCatalog #G3272-1KG
Sodium hydroxideSigma-aldrichCatalog #306576
prepIT.Q2A kit Solution AGDNA GenotekCatalog #PT-Q2A-384 AG
prepIT.Q2A kit Solution STDNA GenotekCatalog #PT-Q2A-384 ST
OR-100 OraCollectRNA kitDNA GenotekCatalog #OR-100
WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA)New England BiolabsCatalog #M1800L
STEP MATERIALS
Sodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
OR-100 OraCollectRNA kitDNA GenotekCatalog #OR-100
prepIT.Q2A kit Solution STDNA GenotekCatalog #PT-Q2A-384 ST
prepIT.Q2A kit Solution AGDNA GenotekCatalog #PT-Q2A-384 AG
Guanidine hydrochlorideSigma AldrichCatalog #G3272-1KG
Water, nuclease-freeThermo FisherCatalog #R0581
WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA)New England BiolabsCatalog #M1800L
Sodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
Note that our kit will contain:
- PCR tubes preloaded with reagents including LAMP Master Mix and primers recognizing a human control RNA and primers recognizing the SARS-CoV-2 virus. One tube of each is used per assay.
- A tube preloaded with 270uL nuclease-free water
- prepIT.Q2A Reagent Solution AG
- prepIT.Q2A Reagent Solution ST
- DNA Genotek OR-100 OraCollect RNA collection swab kit
There is no need to buy OR-100 swab kit, primers, prepIT Solutions, WarmStart Colorimetric Master Mix, nuclease-free water, guanidine HCl or sodium hydroxide outside of the kit unless you plan to make the kit yourself. We consider this an option for labs that have the know-how since this is not a commercial protocol and is open for use by anyone. Labs wanting to make their own kits can follow the Optional Step 1. in our protocol.
Note also that the primer sets can be changed as we learn more about how the virus evolves. The main innovation in this kit is the use of available components in a new way to make the test easy, fast cheap and doable with minimal equipment.
Protocol materials
Sodium hydroxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #306576
Materials
Guanidine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272-1KG
In Materials, Materials, Step 1
prepIT.Q2A kit Solution AGDNA GenotekCatalog #PT-Q2A-384 AG
In Materials, Materials, Step 3
Sodium Hydroxide 1NMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2770-100ml
In Materials, Materials, Materials and 2 steps
primers
Materials
OR-100 OraCollectRNA kitDNA GenotekCatalog #OR-100
In Materials, Materials, Step 2
WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA)New England BiolabsCatalog #M1800L
In Materials, Materials, Step 1
Water, nuclease-freeThermo FisherCatalog #R0581
In Materials, Materials, Step 1
prepIT.Q2A kit Solution STDNA GenotekCatalog #PT-Q2A-384 ST
In Materials, Materials, Step 3
Safety warnings
Use lab coat, gloves and safety glasse when working in the lab.
Before start
Use PPE appropriate to your lab and the population you are swabbing.
OPTIONAL pre-preparation of Master Mix
OPTIONAL pre-preparation of Master Mix
1h
1h
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:
Equipment
4-way tube rack
NAME
Tube rack capable of holding 15mL tubes
TYPE
USA Scientific
BRAND
2304-8044
SKU
LINK
Equipment
0.2 ML PCR PULL-APART 8-TUBE STRIPS, ATTACHED INDIVIDUAL DOME CAPS
NAME
0.2mL PCR tube strips
TYPE
TempAssure
BRAND
1402-2900
SKU
LINK
Equipment
Floating Test Tube Rack
NAME
PCR tube floating foam rack
TYPE
Jaece
BRAND
1201U03
SKU
LINK
Equipment
Disposable polystyrene weighing dishes
NAME
Weigh boat 40mm x 40 mm
TYPE
Sigma
BRAND
Z154873
SKU
LINK
Equipment
Set of micropipettes with rack: 100-1000 µl, 20-200 µl, 2-20 µl, and 0.5-10 µl
NAME
Pipettor set
TYPE
Pipetman
BRAND
QP-1001-07
SKU
LINK
Can use equivalent Pipettors
SPECIFICATIONS
Equipment
80-place tube rack
NAME
1.5mL microfuge tube rack
TYPE
USA Scientific
BRAND
2380-1008
SKU
LINK
Rack that holds 1.5mL microfuge tubes
SPECIFICATIONS
Equipment
TipOne Pipette tips in racks, yellow 200uL
NAME
200uL Pipette tips in racks
TYPE
TipOne
BRAND
1111-0816
SKU
LINK
Equipment
TipOne Pipette tips in racks, natural 10uL
NAME
Pipette tips in racks, natural, 10uL
TYPE
TipOne
BRAND
1111-3810
SKU
LINK
Equipment
TipOne Pipette tips in racks, blue graduated 1000uL
NAME
Pipette tips in racks, 1000uL
TYPE
TipOne
BRAND
1111-2831
SKU
LINK
Equipment
1.5mL microcentrifuge tube, sterile
NAME
1.5mL sterile microfuge tube
TYPE
Seal-Rite
BRAND
1615-5510
SKU
LINK
Equipment
Adventurer™ Analytical Balances
NAME
Analytical balance
TYPE
Ohaus
BRAND
30100600
SKU
LINK
Equipment
Cellstar 15 mL polypropylene conical screw cap centrifuge tube
NAME
Sterile conical screw cap centrifuge tube, 15 mL
TYPE
GREINER BIO-ONE
BRAND
5618-8271
SKU
LINK
Guanidine hydrochlorideSigma AldrichCatalog #G3272-1KG
WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA)Sigma AldrichCatalog #M1800L
Sodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
Water, nuclease-freeSigma AldrichCatalog #R0581
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 CGCTATTAACTATTAACG
For 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 100 micromolar (µM) . 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 -20 °C until ready to use them.
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 100 µL 10x Primer Mix you must add
16 µL BIP
16 µL FIP
2 µL F3
2 µL B3
8 µL LoopF
8 µL LoopB
48 µL nuclease free water
Note 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 -20 °C until ready to use.
Prepare 1M Guanidine HCl
To make 1 Molarity (M) Guanidine HCL , use the weigh boat and analytical balance to weigh out 955 mg Guanidine HCl . Place in a 15mL conical tube in rack. Using the 10mL serological pipette and pipette controller (NEVER pipette by mouth) add 10 mL nuclease free water 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.
Equipment
SCILOGEX Pipette Controllers
NAME
Pipette Controller 10mL
TYPE
SCILOGEX
BRAND
740300029999
SKU
LINK
Equipment
Serological pipette, 10mL
NAME
10ml serological pipette, sterile, individually wrapped
TYPE
USA Scientific
BRAND
1071-0810
SKU
LINK
Equipment
PLASTIC PH TEST STRIPS, UNIVERSAL APPLICATION (PH 0.0-14.0, 0.5 PH INTERVALS), 100 STRIPS
NAME
pH test strips, 0-14
TYPE
LabRat
BRAND
pH 0-14
SKU
LINK
Sodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
Prepare and Aliquot the LAMP Master Mix
Place a 1.5mL tube in the 1.5mL tube rack and using the pipettors add the following for 1mL Master Mix with Covid Primers:
500 µL NEB 2x Colorimetric LAMP Master Mix
100 µL N2 Primer Mix
100 µL E1 primer mix 1
100 µL 1M Guanidine HCl
50 µL nuclease free water
Place PCR tubes in rack. Using the p-20 pipettor and tips, dispense the Master Mix into 0.2mL PCR tubes in the amount of 19 µL per tube
Store frozen at -20 °C until use.
Test Protocol: Sample Collection
Test Protocol: Sample Collection
Using the DNA Genotek OR-100 (aka ORE-100) swab kit for RNA collection
In 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.
OR-100 OraCollectRNA kitSigma AldrichCatalog #OR-100
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.
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.
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.
Shake the tube vigorously for five seconds.
Store at Room temperature until ready to assay.
Test Protocol: Sample Assay
Test Protocol: Sample Assay
19m
19m
Assaying the sample
In 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 primers
Tube containing 270 µL nuclease-free molecular biology grade water
prepIT.Q2A kit Solution STSigma AldrichCatalog #PT-Q2A-384 ST
and
prepIT.Q2A kit Solution AGSigma AldrichCatalog #PT-Q2A-384 AG
A way to heat the tubes to 68 °C for 00:30:00
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.
Equipment
Precision Lid for Anova Precision Cooker
NAME
lid for pot
TYPE
Anova
BRAND
6371115
SKU
LINK
on top of an ordinary kitchen pot, total cost less than $150.
Equipment
Sous Vide - Precision Cooker Nano
NAME
Waterbath heater
TYPE
Anova
BRAND
6238306
SKU
LINK
Other stuff you will need will be:
Equipment
Set of micropipettes with rack: 100-1000 µl, 20-200 µl, 2-20 µl, and 0.5-10 µl
NAME
Pipettor set
TYPE
Pipetman
BRAND
QP-1001-07
SKU
LINK
Can use equivalent Pipettors
SPECIFICATIONS
Equipment
10uL Filter tips
NAME
Pipette tips in racks, natural, 10uL
TYPE
TipOne
BRAND
1121-2710
SKU
LINK
Equipment
20uL filter tips
NAME
Pipette tips in racks, 20uL
TYPE
TipOne
BRAND
1123-1710
SKU
LINK
Equipment
200uL filter tips
NAME
Pipette tips in racks, 200uL
TYPE
TipOne
BRAND
1120-8710
SKU
LINK
Equipment
80 place 1.5 mL microfuge tube rack
NAME
Tube rack
TYPE
BestRack
BRAND
2380-1008
SKU
LINK
Equipment
Compact polypropylene PCR tube rack
NAME
PCR tube rack
TYPE
USA Scientific
BRAND
2396-5048
SKU
LINK
Turn on sous vide heater and set to 68 °C .
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.
1m
Using the P-20 pipettor and tips, add 10 µL prepIT.Q2A Solution AG
1m
Using the P-20 pipettor and tips, add 20 µL prepIT.Q2A Solution ST
1m
Pipette up and down 12 times to mix.
1m
Incubate tube Room temperature for 00:15:00
A precipitate will form and settle to the tube bottom, leaving a clear aqueous layer on top.
15m
Using the P-200 pipettor and tips, transfer 30 µL 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.
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 1 µL of the working dilution from step 3.6 to each PCR tube.
Place the PCR tubes in a floating rack in the 68 °C sous vide waterbath and incubate for 00:30:00
Assay Readout
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.