Sep 10, 2020

Public workspaceRosbash/Janelia StickLAMP Protocol V.2

DOI
dx.doi.org/10.17504/protocols.io.bk89kzz6
  • 1Brandeis University;
  • 2Janelia Research Campus
  • XPRIZE Rapid Covid Testing
Albert Yu
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DOI: dx.doi.org/10.17504/protocols.io.bk89kzz6
Protocol Citation: Albert Yu, Timothy A Brown, Jasmine Quynh Le, Kristina Galatsis, Michael Rosbash 2020. Rosbash/Janelia StickLAMP Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk89kzz6Version created by Albert Yu
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 10, 2020
Last Modified: September 10, 2020
Protocol Integer ID: 41985
Abstract
A protocol for the detection of SARS-CoV-2 from saliva samples featuring a rapid purification step and a high-contrast colorimetric readout. Saliva is first inactivated using a 100x inactivation reagent consisting of 2.5M TCEP, 100 mM EDTA, 1.2N NaOH solution diluted to approximately 1x final concentration and heated to 95C for 5 minutes. RNA is rapidly purified and concentrated with magnetic beads in a PEG/NaCl-based buffer using a 3D-printed magnetic stick that enables selective separation of beads without carryover of saliva contaminants. Beads are eluted directly into an RT-LAMP reaction mix, which uses a novel high contrast dye that turns from purple to clear when acidified by nucleic acid amplification products that enables unambiguous identification of successful amplification. This protocol is sensitive down to 1 copy/µl of SARS-CoV-2 in 300 µl of saliva. This degree of sensitivity enables faithful detection of SARS-CoV-2 even in pooled samples.
Materials
MATERIALS
ReagentNaClSigma AldrichCatalog #53014
ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1
ReagentSARS-CoV-2 Master Mix
ReagentActin Master Mix
Reagent100x Inactivation Reagent
ReagentBead Mix
ReagentMagnetic Tips
ReagentHeat Block at 65C
ReagentHeat Block at 95C
ReagentMagnetic Stick
STEP MATERIALS
ReagentMagnetic Tips
ReagentNaClSigma AldrichCatalog #53014
ReagentSARS-CoV-2 Master Mix
ReagentActin Master Mix
ReagentWater
ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1
100x Inactivation Reagent
2.5M TCEP
150mM EDTA
1.2N NaOH

SARS-CoV-2/Actin Master Mix
12.5µl SARS-CoV-2/Actin Buffer/Dye/Primer Mix (Currently only available from us)
0.5µl WarmStart RTx NEB M0380L
1µl Bst2.0 NEB M0537L
11µl H2O

Primers used
See https://docs.google.com/spreadsheets/d/11n-9754VqtsXszTC2tUxFq-_gKIGgjPL-KtKqXVevH4/edit#gid=0

Bead Mix
See https://ethanomics.files.wordpress.com/2012/08/serapure_v2-2.pdf with 300µl beads instead of 1000µl


Protocol materials
ReagentActin Master Mix
ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1
ReagentSARS-CoV-2 Master Mix
ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1
ReagentMagnetic Tips
Reagent100x Inactivation Reagent
ReagentHeat Block at 65C
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentSARS-CoV-2 Master Mix
ReagentWater
ReagentActin Master Mix
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentBead Mix
ReagentMagnetic Tips
ReagentHeat Block at 95C
ReagentMagnetic Stick
Reagent100x Inactivation Reagent
ReagentSARS-CoV-2 Master Mix
ReagentActin Master Mix
ReagentBead Mix
ReagentMagnetic Tips
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentWater
ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1
Safety warnings
Do not open up PCR tubes after amplification.


Before start
Prepare:

Saliva collection kit (2.0ml Tube and funnel provided by us, or your own saliva collection device from standard labware, such as 1.5ml, 5ml, 15ml, or 50ml tubes. Saliva samples >1ml will likely have to be subsampled)
Magnetic stick
1 magnetic tip per sample
Bead mix: Let bead mix come to room temperature for 20 minutes prior to use, and ensure beads are suspended in solution by vortexing or pipetting up and down
130mM NaCl
SARS-CoV-2 Master mix
Actin Master mix

Saliva Collection
Saliva Collection
Instruct patient to avoid food, drink, toothbrushing, and nasal sprays for a minimum of Duration00:30:00 prior to sample collection



Begin pooling saliva in your mouth. Saliva production can be stimulated by thinking about food, or about the saliva collection itself.
Gently expel saliva into the funnel, tapping to collect in the tube, until amount of saliva is approximately flush with the base of the funnel Amount750 µL Approximately




Inactivation
Inactivation
5m
5m
Add inactivation reagent to approximately 1x final concentration. Reaction is tolerant of between 0.7x to 2x final concentration.Amount7.5 µL Approximately
Reagent100x Inactivation Reagent


Invert 40 times to mix
Heat tube to approximately Temperature95 °C for Duration00:05:00 . Viral RNA release is similar between 93-98C. Use tube clip to prevent popping.
Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK
Any heat block will suffice
SPECIFICATIONS





5m
Remove tube from heat and let rest at TemperatureRoom temperature for at least Duration00:03:00 OR TemperatureOn ice for at least Duration00:00:30 .



While tube is resting, aliquot Amount25 µL SARS-CoV-2 mastermix and Amount25 µL Actin mastermix to separate wells of PCR strip tube, 96-well plate, or 1.5ml tube per sample TemperatureOn ice .

Per run, prepare two additional Amount25 µL SARS-CoV-2 mastermixes for positive and negative controls.


ReagentSARS-CoV-2 Master Mix

ReagentActin Master Mix



Step case

If pooling
From 1 to 10 steps

Prepare one 25ul SARS-CoV-2 reaction and one 25ul Actin reaction per 5 samples
Add approximately 0.7x volumes of bead mix - Amount525 µL Approximately . Sample is tolerant of between 0.7x-1.2x volumes of bead mix. Pipette up and down to mix.
ReagentBead Mix


Step case

If pooling
9 steps

Remove 60ul of inactivated saliva from 5 samples and add to a single tube, for a total of 300µl. Add 210µl bead mix to pooled tube.
Let stand at TemperatureRoom temperature for Duration00:03:00

10m
Cap magnetic stick with a clean tip and dip in bead/sample mix for Duration00:02:00 , dipping up and down 5 times every Duration00:00:30 . Meanwhile, prepare Amount500 µL 130mM NaCl in a separate 1.5ml or 2ml tube.

Equipment
Magnetic Stick
NAME
Rosbash/Brown
BRAND
None
SKU
Magnetic stick used for bead purifications
SPECIFICATIONS

ReagentMagnetic Tips

ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014


2m
Remove magnetic stick from sample and swirl in clean 130mM NaCl solution for Duration00:00:05 . Discard NaCl solution.

5s
Remove magnetic stick from wash sample and place in SARS-CoV-2 mix for Duration00:00:30

30s
Remove magnetic stick from SARS-CoV-2 mix and place in Actin mix for Duration00:00:30

30s
Add Amount5 µL water to additional SARS-CoV-2 Mix (negative control) and Amount5 µL synthetic Twist SARS-CoV-2 positive RNA control to additional SARS-CoV-2 Mix, prepared in Step 8.
ReagentWater


ReagentTwist synthetic SARS-CoV-2 RNA controlTwist BioscienceCatalog #Mt007544.1


Cap tubes and place on Temperature65 °C heating apparatus for Duration00:40:00 .

If using a thermal cycler, run with the following program:

65C for 40 minutes
4C indefinitely
Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK
Any heat block will suffice
SPECIFICATIONS


40m
Remove tubes from heating apparatus and examine color change.


If a positive sample is found when pooling, re-test pooled samples individually.