RT-LAMP is a Loop-Mediated Isothermal Amplification (LAMP) assay that uses a reverse transcription enzyme.
LAMP is a technique that rapidly amplifies DNA. RT-LAMP converts RNA to DNA, and then amplifies the DNA present. RT-LAMP is therefore a reverse transcription LAMP.
What is RT-LAMP used for?
RT-LAMP can amplify and analyze genetic material, and has been used to rapidly detect the COVID-19 virus in samples. RT-LAMP offers quicker analysis of genetic material than traditional PCR, and can be used in point-of-care settings.
LAMP, alongside quantitative PCR (qPCR) and next generation sequencing (NGS) is widely used in pathogen screening. RT-LAMP extends LAMP's uses to detecting RNA target molecules, such as viral RNA.
How does RT-LAMP work?
RT-LAMP relies on auto cycling strand displacement DNA synthesis, and is carried out by three kinds of molecules:
- A reverse transcriptase that converts RNA to DNA, e.g. HIV-1 reverse transcriptase or Bst polymerase, Large Fragment (part of the Bacillus stearothermophilus DNA polymerase).
- A DNA polymerase, with high strand displacement activity, that amplifies DNA in the presence of primers with complementary sequences.
- Six primers: two inner loop primers, two loop primers, and two outer primers. The primers are tailored to the reaction, and the sequence of the target molecule.
Using these primers, RT-LAMP can recognise the target sequence and achieve high specificity. Primer recognition of the target sequence results in a colorimetric reaction, using indicator dyes. These dyes change color based on the amount of certain chemicals that are formed by the DNA produced in the reaction.
What are the benefits of RT-LAMP technology?
RT-LAMP offers several benefits compared to other techniques, including:
- Faster amplification of genetic material compared to PCR.
- Higher specificity and sensitivity.
- No need for a thermal cycler, due to the isothermal conditions.
- Higher amplification efficiency, as there is no need to wait for thermal changes.
- Greater amplification and enrichment compared to PCR. Optimized Bst polymerase results in amplification of up to 109, which is comparable to 30 cycles of PCR, allowing rapid detection of a target sequence.
- Greater resistance to inhibitors in complex samples compared to PCR due to the different polymerase enzyme used.
However, there are some limitations of RT-LAMP. Studies of COVID-19 RT-LAMP protocols suggest cross contamination is a common issue, although it wasn’t found to affect the correct identification of a positive or negative result. Properly sealed well-plates, and the use of certain enzymes may help prevent cross-contamination. Recent studies have suggested other solutions, such as using oligonucleotides as a 'switch' to prevent non-specific amplification of non-SARS-CoV-2 RNA.
In addition, although RT-LAMP has been shown to be as accurate as RT-qPCR at detecting the COVID-19 virus up to the 9th day of symptom onset, beyond this, its accuracy falls.
Magnetic beads can be used alongside RT-LAMP techniques to efficiently isolate and amplify the RNA present. Concentrating the genetic material in the sample with magnetic beads enables RT-LAMP to be carried out with low levels of the target molecule. Bead-LAMP is quick, and nears the sensitivity of gold-standard PCR tests. Real-time detection of the target molecule can be reported with a fluorescent or colorimetric signal.
Bead-LAMP protocols include the following steps:
- Sample lysis
- Preparation of the reaction mix
- Bead-based purification with a magnetic rack
- Bead-LAMP reaction
Find out more about how magnetic bead RNA purification works
RNA purification is vital for a range of applications, including vaccine development and clinical diagnostic testing. Modern magnetic separators have made these tasks even easier, with larger magnetic separation racks accommodating up to 10 liters. Learn more about how magnetic separation works here.