Loop-Mediated Isothermal Amplification (LAMP) is a popular molecular detection technique that was developed in 2000 and uses isothermal conditions for multiplication without the need for a thermocycler. The autocycling strand displacement-based LAMP approach is a two-step process. To create a dumbbell-shaped structure, four primers are first used. Second, the two inner primers are employed to self-prime DNA synthesis in subsequent amplification cycles, using this structure as a template. Cauliflower-like formations made up of an increasing number of concatenated amplicons and stem-loop DNA of various lengths are the results of the LAMP process, which is conducted entirely under isothermal conditions.
Discovery of LAMP
A Japanese business called Eiken Chemical Co., Ltd. created the loop-mediated isothermal amplification of DNA (LAMP) technique in 1998, which got rid of some of the issues with PCR [8]. The method is very specific and can produce up to a billion copies of amplified DNA in less than an hour, as opposed to the PCR’s yield of a million copies. Without sophisticated lab equipment, isothermal amplification can be carried out in a water bath or a dry block heater. Because LAMP uses many primers (four to six), it has a high specificity and can differentiate up to eight distinct sites on the DNA template, whereas normal PCR only uses two. This is another novel feature of LAMP.
Crucial step in LAMP
One crucial component that ensures the proper advancement of the LAMP reaction is the primer design step. It is necessary to optimize a number of primer pairs concerning concentration, nucleotide pair position, and distance between DNA sections. Primers must not form a stable double-strand structure and must have a single-strand structure at 60–65 °C. The interactions between primers can be increased by using a greater number of primers to amplify the same sequence. LAMP primer design can be done with online programs like Premier Biosoft, LAMP Designer Optigene, or PrimerExplorer. Additionally, depending on the LAMP aim, selecting the primers necessitates pre-analysis of the variation of several genomic sequences in the targeted species. In the absence of such data, the time and effort required for a standard LAMP application is significantly increased since sequencing is required to identify the species’ variation of a particular gene.
Primers in LAMP
The primer pairs utilized in LAMP are as follows: the exterior primers, forward primer (F3) and backward primer (B3); the internal primers, forward internal primer (FIP) and backward internal primer (BIP); and the optional loop primers, loop primer forward (FL) and loop primer backward (BL). Long (45–49 bp), the internal primers complement two far-off places on the template (the sense strand and the antisense strand). Because they are shorter (21–24 bp) and added to the reaction mixture at lower concentrations, the exterior primers bind to the template more slowly than the internal primers. The Bst DNA polymerase, which exhibits a strong strand displacement activity at 60–65 °C, combines with the internal and exterior primers, both forward and backward, to form a DNA structure that resembles a dumbbell.
LAMP METHOD
In a DNA pattern, the internal primer of FIP will bind to the pattern strand’s F2-complementing region and start the complementing strand. A dumbbell-shaped DNA structure will be produced as a result of the strand length being extended by the placement of the F3 and B3 external primers with the aid of the Bst (Bacillus stearothermophilus) DNA polymerase enzyme. The LAMP will be initiated by this DNA stem-loop structure. FIP will bind to the DNA’s stem-loop structure and start the synthesis of a successor strand, which will result in a stem-loop intermitted DNA structure with an inverse copy of the target sequence in its stem-loop region that was created by the BIP primer at the other end of the gene. The exterior loop in the structure formed by the released strand serves as the BIP polymer’s pattern. Lastly, DNA in the shape of a dumbbell is created, which will serve as the LAMP cycle’s step material. Heat denaturation following multiplication will not be necessary since oligonucleotide probes tailored to these structures can be used in hybridization. As a result, every stage—from multiplication to detection—could be completed at the same temperature. Stem-loop DNAs with several inverted sequences of the target DNA and a cauliflower-like structure with many loops are the result.
Applications of LAMP
The Loop Mediated Isothermal Amplification method has been used to identify and differentiate pathogenic microorganisms, such as Salmonella species, Nocardia spp., Pseudomonas fluorescens, Staphylococcus aureus, Helicobacter pylori, Mycobacterium tuberculosis, and several other bacteria and medically significant viruses.
Advantages of LAMP
Among the benefits of this approach are
- LAMP can be performed with portable and affordable equipment, utilizing a thermoblock for the necessary temperature without requiring electrophoretic methods for diagnosis. Its primary advantage is the quick, easy, on-the-spot diagnosis.
- The reaction products consist of hairpin-shaped DNAs and cauliflower-like structures formed by annealing repetitive inverse sequences, easily observable under optical microscopes for selective detection.
- LAMP is highly specific, identifying six gene areas with four primers at the start and four others during the reaction. The technique requires only primers, DNA polymerase, and a reaction mixture and can be done in thermal blocks or warm water baths without a thermocycler. The enzyme used is a DNA polymerase with strand displacement activity, enabling significant DNA amplification from few copies.
- Detection does not require electrophoresis; instead, turbidity from gene multiplication and magnesium pyrophosphate precipitates can indicate reaction success and even amplify RNA sequences through inverse copying.
Limitations
Some of its limitations that limit its popularity among researchers include the complexity of the mechanism of this method, the lack of commercial kits based on the LAMP technique, the complexity of multiple primer designs for the multiplication of new gene regions, and the difficulty of choosing the appropriate regions in the gene sequence for efficient primer design. It is also less common than the PCR-based method.
References
Soroka, M., Wasowicz, B., & Rymaszewska, A. (2021). Loop-mediated isothermal amplification (LAMP): the better sibling of PCR?. Cells, 10(8), 1931.
Lee, P. L. (2017). DNA amplification in the field: move over PCR, here comes LAMP.
Keikha, M. (2018). LAMP method as one of the best candidates for replacing with PCR method. The Malaysian Journal of Medical Sciences: MJMS, 25(1), 121.
Khan, M., Wang, R., Li, B., Liu, P., Weng, Q., & Chen, Q. (2018). Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Alternaria solani. Frontiers in microbiology, 9, 2089.
Fakruddin, M. D. (2011). Loop mediated isothermal amplification (LAMP)–an alternative to polymerase chain reaction (PCR). Bangladesh Res Pub J, 5(4), 425-439.