RTLAMP > About LAMP
LAMP stands for loop-mediated isothermal amplification reaction. It’s a mouthful, but what it means is just a biochemical reaction that multiplies DNA. The DNA amplification part is what LAMP and PCR have in common, but then the methods start to differ. The LAMP reaction proceeds in a special way that involves the formation of a “loop” on the molecular level. Once the loop is formed, amplification starts. The amplification is much, much faster than PCR and produces more DNA as well. For that reason, LAMP is sometimes described as an “explosive” amplification reaction.
The amplification of DNA is specific. It can only start when the reaction mix contains short pieces of DNA that match the DNA of the sample. We can make the reaction specific to SARS-CoV-2 virus by using short pieces of DNA called “primers” that are designed to match the genetic information of the virus. Then, you have a reaction mix that starts the LAMP reaction when virus is present in the sample, but it can never start when virus is not present in the sample. This way you have a confirmation of whether or not a sample from a certain person contained the virus – a doctor can use this information to diagnose a person with covid-19.
So far we’ve been talking about DNA. But you may know that SARS-CoV-2 is an RNA virus, that means it has RNA as it’s carrier of genetic information. This is true, but we can still work with it as if it were DNA, if we perform another step. That step is where the RT part comes from – it’s called Reverse Transcription. Simply a conversion of RNA to DNA, which is catalysed by another component of the our reactions – the reverse transcriptase. RT-LAMP is thus able to detect RNA in the sample, and deliver a result based on the amplification of DNA.
We’ve just established that RT-LAMP is a reaction that is capable of triggering the multiplication of DNA, when a specific RNA sequence is present in a sample. But how can we possibly tell if this has happened in a specific test tube? LAMP is such an explosive amplification reaction, that the amount of DNA created by it rapidly shifts the chemical balance of the reaction solution. We can use indicator dyes, that change their colour based on the amount of certain chemical byproducts that are formed by the DNA amplification. A biochemical reaction so powerful, that you can see it’s result with the naked eye!
With a simple, fast and relatively cheap step of concentrating the sample with the use of magnetic beads, we can fit several times the volume of sample into a reaction! This enables the LAMP reactions to “kick off” even with samples that have a very low viral load and would be otherwise undetectable with LAMP or antigen tests. With bead-LAMP, one can approach the sensitivity of the gold-standard PCR tests.
Some RT-LAMP reactions only work with extracted RNA as a sample input. Not ours. It was developed to be compatible with a wide variety of sample inputs, and without the need to do an RNA extraction on these samples. Our RT-LAMP protocols work with swabs in popular swab media and with saliva and gargle. Gargle self-sampling has the best balance of sensitivity, safety, ease of use and comfort. Samples are then inactivated to kill the virus and preserve the RNA for detection by a very short incubation at 95 °C for five minutes in the presence of an inactivation solution. It should be noted that new transfter media that inactivate samples instantly are usually incompatible with RT-LAMP since they contain aggresive chemicals inhibiting the reaction itself.
For result readout, our protocols rely on a visual detection of two different by-products of DNA amplification. For our RT-LAMP protocol, we use a drop in the free magnesium concentration, which is visualised with the help of hydroxynaphtol blue dye by a colour change from purple to sky blue. For bead-LAMP, we can use the colour change caused by the drop of the pH in the sample as a result of DNA amplification. Then, the reaction turns from red to yellow with the help of a dye called phenol red. We can’t use this colour change in normal RT-LAMP, because then the reactions would not be compatible with different sample inputs. We can use it for bead-LAMP though, because we perform the short step of purifying the RNA on magnetic beads, thereby getting rid of the sample which can mess up the pH.
Our RT-LAMP reactions are optimised to not give false positives and to fetch the maximum sensitivity possible. For more information on the sensitivity and specifity, read our manuscript on bioRxiv.
A perfect test is useless when it can’t be deployed on a massive scale. This is why we’ve kept easy access a key component in developing our LAMP assays. Our protocols are public and up to date on our website, and open access protocols have been developed to use the wild type, non patented enzymes BstLF and HIV-RT. This enables anyone around the world to produce them without needing to pay licensing fees or get permission from any company. Only this way can we prevent supply chain issues that have hampered mass testing from the onset of the pandemic. No more running out of one proprietary component – everything is public and available for anyone to synthesise.
We provide protocols for RT-LAMP and bead-LAMP reactions. You can choose if you want to order most of the reagents and enzymes from companies, or if you want to express and purify your own enzymes, or make your own buffers. Protocols for both of these approaches are available on this website.
For under one Euro for the crude reagent costs of RT-LAMP, the low cost enables massive and rapid testing in almost all parts of the world.
A simple, visual colour switch indicates the change in reaction chemistry caused by the presence of the target SARS-CoV-2 molecule in the reaction mix.
Skipping the step of RNA extraction, no need for a thermocycler, no need for fluorescent detection: RT-LAMP reactions run at a constant temperature of 63 degrees Celsius and can be carried out in incubators, thermoblocks or even water baths.
Skipping the step of RNA extraction, no need for a thermocycler, no need for fluorescent detection: RT-LAMP reactions run at a constant temperature of 63 degrees Celsius and can be carried out in incubators, thermoblocks or even water baths.
We set out to develop a test anyone can use. Our open access protocols utilise open-access enzymes that any molecular biology lab can produce at low cost, circumventing reliance on large companies and bringing down the price per test to incredibly small numbers. Buffer compositions are non-proprietary and public.
Gargling has shown to be an effective, comfortable self sampling method for SARS-CoV-2 testing. The use of this self-sampling method decreases the workload of medical personnel, is more comfortable and has proven to be reliable.
RT-LAMP can be done at home with just a few tubes and a pipette, or in a clinical, high-throughput laboratory with liquid handling robotics, ovens and automatic plate readers. Scalability is a key concern for any real-world impact, and this method can be easily automated and adapted in high-tech clinical praxis.
It doesn’t matter if you have swabs in viral transport medium, universal transport medium or saline, gargle in HBSS, saline or water, RT-LAMP, and especially beadLAMP are compatible with a wide variety of sample inputs.
LAMP technology, in and of itself, is very sensitive to contamination. We have implemented a known contamination prevention system into our reactions, based on dUTP and a thermolabile UDG enzyme. This assures that false positives are at a minimum and increases comfort of lab workers.
We need diverse tools to fight a pandemic of this scale. We cannot say that LAMP is the best test in all cases, and flattening the curve will most likely require a complex strategy utilising all of these major test types. You can find a concise comparison of these technologies, how they work and what they differ in.
Viral RNA
Amplification of DNA which is visualised on a real-time fluorescence measurement.
Diagnostic labs
Very high
High
Swab preferred, RNA extraction preferred
2 – 4 hours
Viral RNA
Amplification of DNA which is visualised by a colour change in the reaction tubes.
Diagnostic labs, doctor’s offices, at home.
Moderate to high
Low
Gargle, swab, saliva, no RNA extraction
30 – 45 minutes
Viral surface proteins
Antibody binds to a protein on the surface of the virus and is visualised on a lateral flow strip.
Diagnostic labs, doctor’s offices, at home.
Low to moderate
Low to moderate
Swab
5 – 30 minutes
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