Sep 09, 2020
FloodLAMP Inactivation Protocol v3.1
- Randy True1
- 1FloodLAMP Biotechnologies PBC
- FloodLAMP
- XPRIZE Rapid Covid Testing
Protocol Citation: Randy True 2020. FloodLAMP Inactivation Protocol v3.1. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5nky5e
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 08, 2020
Last Modified: September 09, 2020
Protocol Integer ID: 41870
Abstract
The inactivation protocol uses a chemical plus heat to break open cells (including virus, if present) and preserve the RNA. The inactivation can be done outside a lab setting, at the point of sample collection or drop-off. This is our preferred mode, and we refer to these as "inactivation stations". Prior to inactivation, the samples may contain live, contagious virus, so it is crucial that personnel use proper PPE and safe handling procedures. Many current community screening efforts are performing non-lab based sample processing (for example, Dave O'Connor and Chris Mason), and we have followed their examples with the use of splash guards in addition to PPE.
After inactivation, samples should be refrigerated or stored on ice before same-day processing through the assay.
On our website are our protocols in worksheet form as we use in the lab. This and more information will be live soon.
Guidelines
Individuals are responsible for the chemical and bio safety training to safely complete this protocol. Be careful of aerating samples pre-inactivation. The inactivation solution should also be handled cautiously.
Materials
MATERIALS
Sodium hydroxideSigma – AldrichCatalog #S8045
UltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038
UltraPure Distilled WaterThermo Fisher ScientificCatalog #10977015
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)Sigma AldrichCatalog #C4706
Sigma 100% EthanolCatalog #E7203
Phosphate Buffered SalineThermofisherCatalog #10010023
Tubes:
- 1.5mL Eppendorf DNA LoBind Tubes ($39 for 250)
- 5mL Eppendorf LoBind with screw cap ($79 for 200)
- 5mL colorless Eppendorf DNA tube with screw cap ($65 for 200)
- 5mL amber Eppendorf DNA tube with screw cap ($85 for 200)
- 5mL Eppendorf DNA tube with snap cap ($58 for 200)
- 30mL Self Stading Tubes - Chubs by Stellar Scientific ($99 for 500)
Reagents:
- Zeptometrix
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 dry heater, get out fozen lab armor, turn on centrifuge, make temp QC tube for each size and volume of tube going through inactivation process
- Safety Procedures: always cover heater with plastic cover, wear appropriate PPE
Sample Preparation
Sample Preparation
2m
2m
For dry nasal swabs add 2.5mL 1xPBS, soak swabs for 2minutes, remove and discard swabs properly
2m
For positive controls, spike selected samples with inactivated virions
Inactivation
Inactivation
20m
20m
Spray all closed tubes with 70% ethanol
Vortex the 100x Inactivation Solution within five minutes of use
Add 100x Inactivation Solution to collection tube, add 50uL per 5mL collected sample
2m
For each sample, vortex for 5 seconds, shake and then vortex for another 5 seconds
Heat the samples at 95C for 8 minutes, be sure to add the QC tube(s) for in situ temperature check
8m
Chill samples using the frozen lab armor for 4 minutes, alteratively on ice
4m
Check caps are tight, then Centrifuge samples at 5K rpm for 4 minutes
4m
Aspirate the clarified samples into new tubes, be sure not to disturb any solids at the bottom of the original tube
2m