protocol with open access reagents
Open access means freedom of information. With this protocol, anyone in the world can make their own RT-LAMP assays on a massive scale, producing most of the reagents themselves. Buffers for our open access assays are non-proprietary and their composition and preparation are listed here. Enzymes that power these reactions can be expressed and purified in any molecular biology lab in the world. A plasmid and four days of work is all it takes to produce enough enzymes for hundreds of thousands of RT-LAMP tests for SARS-CoV-2. The open access approach really enables large scale, decentralised and supply chain independent testing strategies. Universities, biohacker collectives, governmental research institutions and many more are encouraged to take matters into their own hands and establish their own, robust production pipelines all over the world.
Order plasmids for enzyme production. Our plasmids are on Addgene for anyone to use.
Follow the published protocols to express enzymes for lots of reactions.
Test your enzyme batches for activity and whole, assembled RT-LAMP reactions for sensitivity.
Help secure easy, fast, and cheap testing for your community.
RT-LAMP reactions are catalysed by two enzymes, a reverse transcriptase that converts RNA to DNA, and a polymerase, that amplifies DNA in the presence of complementary primers. There are many enzymes that can do this, but non-patented wild-type enzymes best suited for this task are HIV-1 reverse transcriptase (HIV-RT) from the human immunodeficiency virus, and Bst polymerase Large Fragment, from the bacterium Bacillus stearothermophilus. Below, you can find the protocol to express these enzymes in E. coli and how to purify them.
Danube is an inactivation buffer based on the HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) protocol. Danube can be prepared as a 10X solution, enabling a larger sample input. Heat inactivation of samples by adding 1 volume of Danube to 9 volumes of sample and heating the mixture to 95 ºC for 5 minutes results in non-infectious samples with a complete inactivation of RNases.
Reagent | Stock concentration | 10x concentration | Volume to add for 1 ml |
---|---|---|---|
TCEP | 0.5 M | 25 mM | 50 μl |
EDTA | 0.5 M | 10 mM | 20 μl |
Betaine | 5 M | 4.15 M | 830 μl |
Proteinase K | 20 mg/ml | 2 mg/ml | 100 μl |
Final Volume | 1 ml |
Isothermal amplification buffer is a well-known reagent in LAMP reactions. It provides optimal reaction conditions for a variety of polymerases derived from BstLF and the wild-type BstLF enzyme as well. A finished, ready to use 10X buffer can be purchased from New England Biolabs, but its composition is publicly known and the buffer itself is very easy to assemble.
Reagent | Stock concentration | 10x concentration | Volume to add for 1 ml |
---|---|---|---|
Tris hydrochloride | 1.5 M | 200 mM | 133 μl |
Potassium chloride | 3 M | 500 mM | 167 μl |
Ammonium sulfate | 1 M | 100 mM | 100 μl |
Magnesium sulfate | 1 M | 20 mM | 20 μl |
Tween® 20 | 100 % | 1 % | 10 μl |
Nuclease-free water | 570 μl | ||
Final Volume | |||
LAMP requires six primers for optimal functioning. That is six short DNA oligonucleotides to synthesise or order, the names are F3, B3, LF, LB, FIP and BIP. To make things easier, one can mix those in a pre-determined ratio to make a 10X primer mix to use with LAMP. You can make this mix with As1 primers against SARS-CoV-2 genome or against human ACTB transcript. Aliquot and label the finished primer mix with the name, date and name of the person who prepared it. We encourage to test every primer mix you receive for activity against the target template, especially when ordering from vendors you don’t have prior experience with.
As1_F3 CGGTGGACAAATTGTCAC
As1_B3 CTTCTCTGGATTTAACACACTT
As1_LF TTACAAGCTTAAAGAATGTCTGAACACT
As1_LB TTGAATTTAGGTGAAACATTTGTCACG
As1_FIP TCAGCACACAAAGCCAAAAATTTATCTGTGCAAAGGAAATTAAGGAG
As1_BIP TATTGGTGGAGCTAAACTTAAAGCCCTGTACAATCCCTTTGAGTG
ACTB-F3 AGTACCCCATCGAGCACG
ACTB-B3 AGCCTGGATAGCAACGTACA
ACTB-FIP GAGCCACACGCAGCTCATTGTATCACCAACTGGGACGACA
ACTB-BIP CTGAACCCCAAGGCCAACCGGCTGGGGTGTTGAAGGTC
ACTB-LoopF TGTGGTGCCAGATTTTCTCCA
ACTB-LoopB CGAGAAGATGACCCAGATCATGT
Oligo name | Add for 100 μl of mix |
---|---|
F3 | 2 μl |
B3 | 2 μl |
LF | 4 μl |
LB | 4 μl |
FIP | 16 μl |
BIP | 16 μl |
nuclease-free water | 56 μl |
When you have the enzymes, primer mix and the sample inactivation reagent, it’s time to assemble the reactions themselves. Make sure to test your reactions on a known positive sample dilution first to assess the specificity and sensitivity of your assays. Expressing and purifying your own enzymes is not trivial, but the payoff is tremendous. If you are expressing your enzymes and having issues with the resulting reactions, please contact us for help in troubleshooting. Please consider the following before you start:
Reagent | Stock concentration | Final concentration | To add per reaction |
---|---|---|---|
Isothermal amplification buffer | 10X | 1X | 1 μl |
dNTP mix | 25 mM | 1.4 mM | 0.56 μl |
dUTP | 100 mM | 0.7 mM | 0.07 μl |
Magnesium sulfate | 100 mM | 6 mM | 0.6 μl |
As1 primer mix | 10X | 1X | 1 μl |
HNB dye, trisodium salt | 20 mM | 0.12 mM | 0.06 μl |
Syto9 fluorescent dye (optional) | 100 μM | 2 μM | 0.2 μl |
Betaine | 5 M | 0.4 M | 0.8 μl |
HIV-RT enzyme | 0.55 mg/ml | 0.00275 mg/ml | 0.05 μl |
BstLF enzyme | 0.6 mg/ml | 0.02 mg/ml | 0. 33 μl |
Thermolabile UDG enzyme | 1 U/μl | 0.02 U/μl | 0.2 |
Nuclease-free water | to a total of 8 μl | ||
Final Volume | 8 μl | ||
Hydroxynaphtol blue (HNB) is used in RT-LAMP reactions that do not include any purification step. The dye changes colour from purple to sky blue upon presence of target sequence and amplification. This can be visualised with the naked eye, and reactions can be scored directly after taking them out of the heating device.
For people who find the colour change of HNB difficult to see with the naked eye, we recommend the use of a simple web app. Developed by Andrew Straw to increase the colour difference between positives and negatives and make the readout much simpler. Try it out on colorimetry.net
Amplification of DNA in RT-LAMP reactions can also be read out through the use of fluorescent intercalating dyes. These dyes, such as Syto9 or the NEB LAMP dye, enable the user to either monitor the progress of the reaction in real time through the use of a qPCR machine, or end-point detection with a plate reader or a similar fluorescence detection device.
Assembling each master mix for every testing session from scratch is not necessary, however freezing prepared master mixes increases the risk of running into sensitivity or specificity issues. We’ve tested the possible combinations and now we can tell you what you can and can’t premix for easier reaction assembly without compromising on the quality of the tests.
Reagent | Stock concentration | To add per 100 reactions (10µl size) | |
---|---|---|---|
Isothermal amplification buffer | 10X | 100 μl | |
dNTP mix | 25 mM | 56 μl | |
dUTP | 100 mM | 7 μl | |
Magnesium sulfate | 100 mM | 60 μl | |
As1 primer mix | 10X | 100 μl | |
HNB dye | 20 mM | 6 μl | |
Syto9 fluorescent dye (optional) | 100 μM | 20 μl | |
Betaine | 5 M | 80 μl | |
nuclease-free water | 321 μl | ||
total | 750 μl |
Add 7.5 µl of this mix per 1 reaction.
Store at -20 °C.
Reagent | Stock concentration | Amount per reaction | To add per 100 reactions (10µl size) |
---|---|---|---|
HIV-RT enzyme | variable | 75 ng | variable |
BstLF enzyme | variable | 200 ng | variable |
Thermolabile UDG enzyme (NEB) | 1 U/μl | 0.2 U | 20 μl |
storage buffer | to a total of 50 µl | ||
total | 50 µl |
Add 0.5 µl of this mix per 1 reaction.
The volumes for enzymes are listed as “variable” since each enzyme purification yields a different concentration of enzyme. We recommend measuring the yield you get from your purifications and make the calculations yourself, so as to reach 75 ng of reverse transcriptase and 200 ng of DNA polymerase, respectively. Then multiply the volumes for 75 ng and 200 ng of enzyme, respectively, by 100 to fill in the table. Add storage, as described in the enzyme expression and purification protocol on the open access protocol pages, to a total of 50 µl.
Store at -20 °C.
The premixes for open-access RT-LAMP enable you to have two tubes in your -20 °C freezer and to assemble reactions easily using 7.5 µl of LAMP reaction buffer premix and 0.5 µl of LAMP enzyme premix per 10 µl reaction. We’ve observed their stability to be unchanged after 1 week’s storage at -20 °C, but they can most likely last for longer. It is crucial to work quickly with the enzyme mix and to work with it on ice or in the cold.
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