Reverse Transcription Loop-mediated Isothermal Amplification

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a method of amplifying nucleic acids under isothermal conditions where reverse transcriptase is included in the reaction in order to amplify an RNA template. At the beginning of the reaction a primer anneals to the RNA template at 60-65 C and reverse transcriptase activity results in synthesis of cDNA. DNA polymerase with strand displacement produces DNA strands. The primers are designed to contain complementary sequences at both ends so the ends self-anneal and form a dumbbell-like structure. This is the starting structure for LAMP cycling amplification. The final products are a mixture of stem-loop DNAs of various stem lengths and cauliflower-like structures. Multiple loops are formed by annealing between alternately inverted repeats of the target sequence in the same strand. Target specificity is high since four primers are used, which recognize six distinct recognition sequences.

RT-LAMP assays are being developed for in vitro diagnostics as an alternative to quantitative reverse transcription PCR (qRT-PCR) as RT-LAMP is more suitable for low-resource settings and point-of-care (POC) platforms. RT-LAMP has applications in clinical diagnosis and infectious disease surveillance. RT-LAMP amplification can be detected by the naked eye though color change in the reaction tube.

The LAMP reaction can be measured through real-time turbidity with a photometer or turbidimeter. Insoluble magnesium pyrophosphate is produced in the course of the amplification. This method can be used to quantify the initial template. Calcein is a fluorescent molecule that is quenched by manganese ions and will produce fluorescence as the manganese ions bind pyrophosphate ions produced during amplification.

Researchers from Isreal, led by Naama Geva-Zatorsky, developed an RT-LAMP assay to detect coronavirus SARS-COV-2 (COVID-19) from clinical diagnostic swabs without RNA purification. A colorimetric test using RT-LAMP to detect SARS-CoV-2 virus RNA was also published by researchers based in New York, led by Chris Mason. Mammoth Biosciences developed a nucleic acid detection platform called DETECTR which uses RT-LAMP and CRISPR technology (CRISPR-Cas12a) and have demonstrated its ability to detect SARS-CoV-2 in nasopharyngeal or oropharyngeal swabs.