Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by
RNA viruses.[1]
It combines
LAMP[2] DNA-detection with
reverse transcription, making
cDNA from RNA before running the reaction.[3] RT-LAMP does not require
thermal cycles (unlike
PCR) and is performed at a constant temperature between 60 and 65 °C.
RT-LAMP is used to test for the presence of specific RNA-samples of viruses for the specific sequence of the virus, made possible by comparing the sequences against a large external database of references.
Detection of the SARS-CoV2-Virus
The RT-LAMP technique is being supported as a cheaper and easier alternative to
RT-PCR for the early diagnostics of people that are infectious for
COVID-19.[6] There are
open access test designs (including the
recombinant proteins) which makes it legally possible for anyone to produce a test. In contrast to classic rapid tests by
lateral flow, RT-LAMP allows the early diagnosis of the disease by testing the
viral RNA. [7]
The tests can be done without previous RNA-isolation, detecting the viruses directly from swabs[8] or from
saliva. [9]
Detection of non-human viruses
One example of use case of RT-LAMP was as an experiment to detect a new
duck Tembusu-like, BYD virus, named after the region,
Baiyangdian, where it was first isolated[10][11][1] Another recent application of this method, was in a 2013 experiment to detect an Akabane virus using RT-LAMP. The experiment, done in China, isolated the virus from aborted calf fetuses.[12]
Detection of body fluids
RT-LAMP is also being used in
Forensic Serology to identify body fluids. Researchers have done experiments to show that this method can effectively identify certain body fluids. Knowing there would be limitations, Su et al, come to the conclusion that RT-LAMP was only able to identify blood.[13][14]
Methodology
Reverse transcription
A specific sequence of the cDNA is detected by 4 LAMP
primers. Two of them are inner primers (FIP and BIP), which serve as base for the
Bst enzyme copy the template into a new DNA. The outer primers(F3 and B3) anneal to the template strand and help the reaction to proceed.
As in the case of
RT-PCR, the RT-LAMP procedure starts by making DNA from the sample RNA. This conversion is made by a
reverse transcriptase, an enzyme derived from
retroviruses capable of making such a conversion.[15] This DNA derived from RNA is called
cDNA, or complementary DNA. The FIP primer is used by the
reverse transcriptase to build a single-strand of copy DNA. The F3 primer binds to this side of the template strand as well, and displaces the previously made copy.
Amplification
This displaced, single-stranded copy is a mixture of target RNA and primers. The primers are designed to have a sequence that binds to the sequence itself, forming a loop.
The BIP primer binds to the other end of this single strand and is used by the
Bst DNA polymerase to build a complementary strand, making double-strand DNA. The F3 primer binds to this end and displaces, once again, this newly generated
single-stranded DNA molecule.
This new single strand that has been released will act as the starting point for the LAMP cycling amplification. This single-stranded DNA has a
dumbbell-like structure as the ends fold and self-bind, forming two loops.
The DNA polymerase and the FIP or BIP primers keep amplifying this strand and the LAMP-reaction product is extended. This cycle can be started from either the forward or backward side of the strand using the appropriate primer. Once this cycle has begun, the strand undergoes self-primed DNA synthesis during the elongation stage of the amplification process. This amplification takes place in less an hour, under isothermal conditions between 60 and 65 °C.
Read out
The read out of RT-LAMP tests is frequently colorimetric. Two of the common ways are based on measuring either
pH or
magnesium ions. The amplification reaction causes pH to lower and Mg2+ levels to drop. This can be perceived by indicators, such as
Phenol red, for pH, and
hydroxynaphthol blue (HNB), for magnesium.[15] Another option is to use
SYBR Green I, a DNA intercalating coloring agent.[16]
Advantages and disadvantages
This method is specifically advantageous because it can all be done quickly in one step. The sample is mixed with the primers, reverse transcriptase and DNA polymerase and the reaction takes place under a constant temperature. The required temperature can be achieved using a simple hot water bath.
PCR requires
thermocycling; RT-LAMP does not, making it more time efficient and very cost effective.[3] This inexpensive and streamlined method can be more readily used in developing countries that do not have access to high tech laboratories.
A disadvantage of this method is generating the sequence specific primers. For each LAMP assay, primers must be specifically designed to be compatible with the target DNA. This can be difficult which discourages researchers from using the LAMP method in their work.[1] There is however, a free software called Primer Explorer, developed by Fujitsu in Japan, which can aid in the selection of these primers.
^Lalli, Matthew A.; Langmade, S Joshua; Chen, Xuhua; Fronick, Catrina C.; Sawyer, Christopher S.; Burcea, Lauren C.; Wilkinson, Michael N.; Fulton, Robert S.; Heinz, Michael; Buchser, William J.; Head, Richard D.; Mitra, Robi D.; Milbrandt, Jeffrey (2020). "Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP".
medRxiv10.1101/2020.05.07.20093542.
^Jiang T, Liu J, Deng YQ, Su JL, Xu LJ, Liu ZH, Li XF, Yu XD, Zhu SY, Gao GF, Qin ED, Qin CF (December 2012). "Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus". Arch. Virol. 157 (12): 2273–80.
doi:
10.1007/s00705-012-1431-7.
PMID22865206.
S2CID15573433.