Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification used as an alternative to polymerase chain reaction (PCR). LAMP has been used as a rapid, accurate, and efficient method for the detection and identification of infections including those that are bacterial, viral, fungal, and parasitic in nature for humans, animals, and plants. Notably, some RNA viruses like HIV-1, Dengue virus, SARS-CoV-2, and Ebola (EBOV), as well as the bacterial disease, meningitis, have been detected through LAMP techniques.

LAMP has widely been used as a rapid, diagnostic tool to ensure timely clinical treatment and prevention of disease outbreaks. The LAMP protocol is incredibly simple; all it requires are at least 4 suitable primers (often 6), a DNA polymerase, and a regular laboratory water bath, heat block, or oven for processing. LAMP can be used as a powerful point of care device and with variations in the technique the availability for high throughput screening has increased.

LAMP techniques work very similar to PCR, but can be completed much faster, in roughly 90 minutes. Some variations of LAMP even have shorter test times, ranging from 30-60 min. LAMP techniques have the ability to amplify just a few copies of DNA to 109 copies, and do not require the use of a thermocycler like PCR. LAMP methods are performed under isothermal conditions at a constant temperature range between 60-65°C. The precision of this technique, coupled with its simplicity and amplification efficiency, make it a suitable, portable field test, even in the most remote settings. LAMP carries the added benefit of efficiently amplifying RNA sequences when used in conjunction with reverse transcription techniques. Additionally, various LAMP master mixes are readily, commercially available.

General LAMP techniques employ the use of bacillus stearothermophilus (Bst), a DNA polymerase, which possesses a high strand-displacement activity. In most LAMP methods 6 different primers are designed to recognize 8 distinct regions on a target gene, and amplification will occur if the primers bind and form the intended product.

Two sets of inner primers are used (forward and backward inner primers) along with two sets of outer primers (forward and backward outer primers). The LAMP reaction is initiated by an inner primer containing sequences of sense and antisense strands of the target DNA. Using the stem-loop areas as a stage, DNA polymerase-mediated strand displacement synthesis repeats the elongation events continually. After, denaturation releases single-stranded DNA (ssDNA) by an outer primer. ssDNA serves as a template for DNA synthesis primed by the second inner and outer primers that will then hybridize together at the other end of the target structure. LAMP results in the formation of a stem-loop DNA structure that are, essentially, inverted repeats of the target DNA or RNA sequence. The LAMP reaction may also be accelerated by the addition of two loop primers (one forward and one backward).

LAMP amplified products can be visualized using agarose gel electrophoresis, turbidity, fluorescence, or via colorimetric means. Upon turbidity, the formation of white precipitate caused by magnesium pyrophosphate, a by-product of the reaction, in the amplified mixture is linearly correlated with the amount of DNA synthesized. Turbidity can be quantitatively measured using a turbidimeter, or examined by the naked eye. For electrophoretic, fluorometric, or colorimetric visualization methods, color changes in amplified products can often be detected through simple observation, then quantified.

Variations of LAMP techniques have been created that couple other research methods for more precise and accurate analysis. To date, only a handful of LAMP assays have been approved for clinical use due to, mainly, a low tolerance to highly variable target sequences, non-specific amplification, and the limitations associated with multiplexing. These variations in the LAMP technique are limited in uniformity as they are most commonly practiced in research, though numerous studies exist that provide evidence of how LAMP has the potential to advance clinical diagnostics.

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