Sep 07, 2020
BW SARS-CoV-2 Laboratory Test
- 1Biology Works LLC
- XPRIZE Rapid Covid Testing
- Biology Works LLC
Protocol Citation: Ivan D Rueda, peter.marx 2020. BW SARS-CoV-2 Laboratory Test. protocols.io https://dx.doi.org/10.17504/protocols.io.bkztkx6n
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 07, 2020
Last Modified: September 07, 2020
Protocol Integer ID: 41747
Disclaimer
This protocol is not approved for clinical use.
Abstract
This protocol is the laboratory-based equivalent of the LAMP assay used for detecting SARS-CoV-2. A similar protocol is performed automatically within the Biology Works LLC at-home device.
Image Attribution
Photograph by Ivan Rueda.
Guidelines
This protocol is not approved at this time by any regulatory body for clinical use. It uses proprietary reagents available from Biology Works LLC and other vendors. Repetitive freezing of primers is not recommended; please store them at 4°C when not in use.
Materials
STEP MATERIALS
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Guanidine HCLMillipore SigmaCatalog #G7294
Guanidine HCLMillipore SigmaCatalog #G7294
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
HyClone WaterGe Life SciencesCatalog #SH30538LS
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Protocol materials
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
HyClone WaterGE HealthcareCatalog #SH30538LS
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
HyClone WaterGE HealthcareCatalog #SH30538LS
Safety warnings
This protocol is not approved at this time by any regulatory body for clinical use. You must follow all safety protocols specified by each vendor, your laboratory, common sense, and regulatory bodies in your geography. All specimens, reagents, and equipment must be handled asceptically. Personal protective equipment, including appropriate gloves, clothing and protective eye ware must be worn at all times.
Before start
Acquire a nasal or saliva sample for use with this assay. The Chai Bio 1-step DNA/RNA Extraction Buffer (Cat. 05210S) has been demonstrated as being compatible with nasal swabs using this protocol. Saliva samples must be heat denatured at 95°C for 10 minutes.
CITATION
Assay
Assay
33m 40s
33m 40s
Thaw reagents to room temperature Room temperature
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
Vortex reagents 3000 rpm 3x
Equipment
Vortexer
NAME
VWR
BRAND
97043-562
SKU
15s
Briefly spin down components in a microcentrifuge 00:00:05
Equipment
Microcentrifuge
NAME
VWR
BRAND
75993-410
SKU
5s
Prepare master mix in a 1.5mL tube with the following sub-steps
Equipment
Seal-Rite 1.5mL Microcentrifugre Tube
NAME
USA Scientific
BRAND
1615-5510
SKU
Add 12.5 µL to tube
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Add .3125 µL to tube
Guanidine HCLMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7294
Add 4 µL to tube
BW SARS-CoV-2 Primer MixBiology Works LLCCatalog #SARS-CoV-2 Primer Mix
Add 0.5 µL to tube
LAMP Fluorescent DyeNew England BiolabsCatalog #B1700AA
Add 2.8 µL to tube
HyClone WaterGE HealthcareCatalog #SH30538LS
Close tube cap
Vortex 3000 rpm 3x
15s
Briefly spin down components in a microcentrifuge 00:00:05
5s
Aliquot out 20uL into a PCR reaction plate
Equipment
Microamp Optical 96-well Reaction Plate
NAME
Thermo-Fisher
BRAND
N8010560
SKU
Dispense 5 µL of your sample solution into the well
Seal plate with film
Equipment
Microamp Optical Adhesive Film
NAME
Thermo-Fisher
BRAND
4311971
SKU
Centrifuge plate in plate centrifuge 500 rcf
Equipment
Centrifuge 5810 R
NAME
Plate Centrifure
TYPE
Eppendorf
BRAND
022625101
SKU
Place plate with reagents and sample into thermo-cycler
Equipment
7500 Real Time PCR System
NAME
ABI
BRAND
4351104
SKU
Run assay on thermo-cycler with the following protocol (specific to the ABI 7500):
1x 25 °C 00:02:00
60x 64.9 °C 00:00:02 and 65 °C 00:00:30
Record measurements
33m
Read results
Citations
S. Bhadra, T. E. Riedel, S. Lakhotia, N. D. Tran, and A. D. Ellington. High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes
10.1101/2020.04.13.039941