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From the get-go, one of our focuses was on enabling accurate SARS-CoV-2 detection on a massive scale. Where supply chains of large multinational corporations failed in enabling affordable, available and robust testing programmes, we hope that a decentralised and open sourced solution might be the cure. This is why we developed our LAMP assays also as open access protocols, without the use of proprietary reagents and supplies, relying entirely on non-patented wild type enzymes. We demonstrate that this approach is comparable in sensitivity to the commercial one and a feasible alternative for many settings.
Just like the commercial protocol, the reactions work through the enzymatics provided by a strand displacing DNA polymerase and a thermostable reverse transcriptase. The features that make our protocol robust, simple and usable in low-resource settings are present in the open-access protocols as well. That includes the UTP/UDG cross contamination prevention system, a visual pH-independent colorimetric readout and the simple 5 minute virus inactivation and lysis step.
This is what powers the reactions
HIV-1 reverse transcriptase
The human immunodeficiency virus 1 (HIV-1) is a retrovirus and the causative agent of AIDS. Retroviruses are a family of RNA viruses who transcribe their genetic information into DNA and then incorporate that DNA into host cells. This transcription activity from RNA to DNA, so called reverse transcription, is an enzymatic reaction immensely useful for many molecular biology applications. In LAMP, the HIV's reverse transcriptase enzyme (HIV-1 RT) serves to transcribe viral RNA into DNA which is then amplified by the Bst polymerase.
Pictued: HIV virions (green) budding from lymphocyte. Photo Credit: C. GoldsmithContent Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus, Public domain, via Wikimedia Commons
Bst DNA polymerase, large fragment
A DNA polymerase for our reactions comes from the bacterium Geobacillus stearothermophilus. This species of bacteria lives at elevated temperatures, thus it's DNA polymerase is able to function even at 60 - 70 degrees, which is the temperature that most LAMP protocols use. It posesses a strand-displacement activity, which means that the polymerase is capable of unwinding double stranded DNA. This removes the need for thermal cycling (like in PCR) for DNA amplification and enables LAMP to be carried out at a single temperature, like in an incubator or a water bath.
Pictued: Bacillus sp. gram stain. Photo credit: Dr. Sahay, CC BY-SA 3.0, via Wikimedia Commons
The open-access LAMP approach can allow laboratories, universities and entire countries to start their own mass testing programmes without the need to rely on diagnostics companies. Doubly as important in a pandemic situation, when supply and demand changes rapidly and commercial supply chains either outright fail to service the needs of large scale testing needs at best and act as price-gouging profiteers in times of crisis at worst. With these protocols, any equipped and established molecular biology laboratory can take things into their own hands and secure the testing needs either for their coworkers, campus or cities. Mere four litres of bacterial culture are enough to perform 100 000 reactions! This is the workflow one usually follows when establishing enzyme production for SARS-CoV-2 detection.
Order reagents
Get the Bst-LF and HIV-1 RT plasmids from Addgene. Get chemicals and primers from suppliers.
Express enzymes
Follow our protocols to express and purify the LAMP enzymes. Perform batch testing on the enzymes.
Test, test, test!
Use our open access protocols to assemble your reaction mixes and perform LAMP tests to beat back the pandemic.
There are already several labs over the world that have successfully expressed the wild type enzymes for use in SARS-CoV-2 detection and are now setting it into practice. Surveillance and screening tests can be especially efficient with this approach, as well as diagnostics in low resource settings.
With rates of covid-19 infections on the rise again, our group decided that developing an in house diagnostic technique to screen members of the lab and those we work with was the best way to ensure safe working conditions. We turned to the RT-LAMP protocol developed at the IMP and IMBA as this offered itself as a rapid, cheap, and reliable method to diagnose covid-19 infections. I found their open source protocol clear and simple to set up, and within a short period of time I had the assay working well with my own reagents, including the BstLF polymerase and HIV-RT enzymes I expressed and purified from plasmids that were provided. In my hands, I found the assay was extremely sensitive, and under lab conditions could detect as few as 100 molecules of RNA template in a standard 10 µL reaction. I am currently in contact with a local diagnostic lab to test the protocol in diagnosing genuine infections using my own proteins and reagents and I can then confidently role this out to members of my lab and department. Thanks to the Pauli group at the IMP for all their help!
What’s important to note is that for any diagnostic purposes, one has to go through the proper legal procedures that are applicable for the given jurisdiction. We do not encourage anyone to use our protocols and pass the results off as diagnostically valid. We can not be held liable for anything on the side of the user of the protocol. All we have are functioning and robust protocols for SARS-CoV-2 detection, and a firm belief that sharing the information on how to perform them correctly can help in a situation like this. This is why we’re putting it out there.
Open access protocols will guide you through the preparation of virus inactivation solution, reaction buffer, LAMP primer mixes and of course the expression and purification of BstLF DNA polymerase and HIV-RT reverse transcriptase. Click on the buttons below to reach out open access RT-LAMP protocol and bead-LAMP protocol.