Sep 09, 2020
FloodLAMP LAMP Assay Protocol v4.1 (0.5ml, Strips, Multichannel)
- Randy True1
- 1FloodLAMP Biotechnologies PBC
- FloodLAMP
- XPRIZE Rapid Covid Testing
Protocol Citation: Randy True 2020. FloodLAMP LAMP Assay Protocol v4.1 (0.5ml, Strips, Multichannel). protocols.io https://dx.doi.org/10.17504/protocols.io.bkvnkw5e
Manuscript citation:
[1] Rabe B, Cepko C. SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification. medXriv preprint 4-28-20 https://www.medrxiv.org/content/10.1101/2020.04.23.20076877v1
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 04, 2020
Last Modified: September 09, 2020
Protocol Integer ID: 41614
Abstract
FloodLAMP uses an assay chemistry developed by Brian Rabe and Prof. Connie Cepko at The Cepko Lab at Harvard Medical School. "Rabe Cepko," as it's known, uses an ultra cheap, streamlined front end (sample inactivation and RNA purification+concentration). This protocol has been chosen by many groups, from pharmaceutical companies to researchers working in the developing world. The same aspects that make it ideal for Africa also make it ideal for America—it's cheap, works well, and can be implemented by any basic chemistry or biology lab.
This assay was validated clinically in May by researchers at Mass General Hospital. In addition, the key amplification reagent (NEB Colorimetric LAMP Master Mix) is the same product used in the FDA-approved test by Color Genomics, who does about 5K tests per day in the SF Bay Area.
Our current preferred version incorporates a transfer of the nucleic acid bound silica in ethanol to the PCR tubes (strips or plates) where the LAMP reaction is carried out. The pellet resuspends easily in the 80% ethanol and—once in the PCR tubes—can be quickly dried on a heat block. Then they are ready for addition of the 1X LAMP Reaction Mix. We have used alternatives to this method, including an elution of the nucleic acid and also a resuspension of the pellet in water or 1X PBS, then addition to the LAMP reaction. With the LAMP Master Mix (NEB 1804), the assay uses 2 prepared solutions: a NaI Binding Solution and the Glass Milk (also called "prepared silica").
On our website are our protocols in worksheet form as we use in the lab. This and more information will be coming soon.
Guidelines
Individuals are responsible for the chemical and bio safety training to safely complete this protocol.
Materials
MATERIALS
Triton X-100Sigma AldrichCatalog #T8787-50ML
Hydrochloric acidSigma – AldrichCatalog #320331-500ML
Sodium hydroxideSigma – AldrichCatalog #S8045
UltraPure Distilled WaterThermo Fisher ScientificCatalog #10977015
UltraPure™ 0.5M EDTA, pH 8.0Thermo FisherCatalog #15575020
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)Sigma AldrichCatalog #C4706
Guanidine hydrochlorideSigma AldrichCatalog #G3272-1KG
Sigma 100% EthanolCatalog #E7203
Sodium Iodide (NaI)SigmaCatalog #793558
Silicon DioxideSpectrum ChemicalsCatalog #SI108
Phosphate Buffered SalineThermofisherCatalog #10010023
10X Primers (As1e and N2 combined)IDT Technologies
Tubes:
- USA Scientific TempAssure 0.2 mL, 8 pieces PCR Tube Strips w/ Optical Flat Cap Strips ($83 for 125 strips)
- 1.5mL Eppendorf DNA LoBind Tubes ($39 for 250)
- 5mL Eppendorf DNA LoBind with screw cap ($79 for 200)
- 5mL Eppendorf with screw cap, amber ($85 for 200)
- 5mL Eppendorf DNA tube with snap cap ($58 for 200)
- 30mL Self Stading Tubes - Chubs from Stellar Scientific ($99 for 500)
Reagents (see website for more information):
- Zeptometrix Inactivated virions (NATSARS(COV2)-ST)
- SARS-CoV-2 RNA Control 1, Twist RNA (102019)
Safety warnings
Both TCEP and EDTA should be handled cautiously as they can cause severe eye damage and are toxic if inhaled. See SDS for TCEP, EDTA, NaOH, HCl and NaI for more safety information.
Before start
- Set Up: turn on heat block to 65C with PCR tube block inside
- Safety Procedures: always wear appropriate PPE
Purification ( .5mL sample)
Purification ( .5mL sample)
14m
14m
Spike selected samples with Twist RNA
1m
For each 8 samples, Add 45uL of glass milk (GM) to 2.25mL of binding solution (BS), vortex glass milk before using, flicking tube to be sure that there is no pellet at the bottomand it's evenly mixed
30s
Add 255uL of BSGM to 500ul samples, while adding BSGM pipette up and down 3x at bottom, 3x in middle, draw from middle. Start 10min timer after last addition.
2m
Vortex samples 3sec, before adding to rocker or rotator. Alternatively shake or vortex every 2min.
10m
Spin down samples for 1min
30s
Express Wash and Transfer
Express Wash and Transfer
24m
24m
Pour the supernatant into a "sample supernatant" 50ml Falcon tube (or other waste)
Gently add 900uL of 80% EtOH, do not disturb the pellet (predraw about 100ul of air so next aspirate removes all liquid)
Aspirate all of the EtOH using the same tip and discard in "ethanol supernatant" 50ml Falcon tube (or other waste)
Add 100uL 80% EtOH, resuspend the pellet and transfer to labeled PCR strip
When all are in PCR strips, with P200 multichannel pipette, transfer 20uL from each to 2nd PCR strip. Cap all strips.
2m
Spin down strips for 1min
1m
Aspirate supernatant with 200uL multichannel pipette, hugging side of tubes away from pellet
1m
Heat strips on heat block set at 65C for 5-10 minutes (open covered with foil), the pellet should look chalky and not wet
10m
LAMP Reaction
LAMP Reaction
40m
40m
Make the ALL MasterMix, two separate tubes for R and AN primers
3m
For each strip of 8, add 66uL dH2O, 22uL of 10X Guanidine, vortex 3s
Add 22uL of the 10x primers (AN for 80% pellet strip and R for 20%), vortex 3s
Add 110uL of LAMP MM, vortex 3s
Add 25uL x n strips + 2uL (27, 52, ...) of the ALL MM to each tube in a new PCR strip, one strip for R and one for AN
1m
With multichannel pipette, add 25uL of ALL MM to each of the pellet strips using the multichannel 200uL pipette (AN for 80% pellet strip and R for 20%), pipet up and down 5x
5m
Cap strip tubes, remove from holder, check caps, flick to mix, snap down
1m
Incubate strip tubes on heat block at 65C for 25min (can check at 20min and 30min)
30m
Observe tube color, should be bright pink. Note if orange tinted (likely pellet was not dry).
Remove strips, let cool for at least 1min before snapping photo.