Reverse Transcription Loop-mediated Isothermal Amplification

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

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

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

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.

See also

• Loop-mediated isothermal amplification

References