Techniques for Evaluation of LAMP Amplicons and their Applications in Molecular Biology

Abstract

Loop-mediated isothermal amplification (LAMP) developed by Notomi et al. (2000) has made it possible to amplify DNA with high specificity, efficiency and rapidity under isothermal conditions. The ultimate products of LAMP are stem-loop structures with several inverted repeats of the target sequence and cauliflower-like patterns with multiple loops shaped by annealing between every other inverted repeats of the amplified target in the similar strand. Because the amplification process in LAMP is achieved by using four to six distinct primers, it is expected to amplify the target region with high selectivity. However, evaluation of reaction accuracy or quantitative inspection make it necessary to append other procedures to scrutinize the amplified products. Hitherto, various techniques such as turbidity assessment in the reaction vessel, post-reaction agarose gel electrophoresis, use of intercalating fluorescent dyes, real-time turbidimetry, addition of cationic polymers to the reaction mixture, polyacrylamide gel-based microchambers, lateral flow dipsticks, fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays and nanoparticle-based colorimetric tests have been utilized for this purpose. In this paper, we reviewed the best-known techniques for evaluation of LAMP amplicons and their applications in molecular biology beside their advantages and deficiencies. Regarding the properties of each technique, the development of innovative prompt, cost-effective and precise molecular detection methods for application in the broad field of cancer research may be feasible.

Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

“…The heat treatment did not interfere with turbidity readouts according to our spiked sera results and the previous report [19]. Several detection platforms were integrated to LAMP such as lateral flow dipstick, enzyme-linked immunosorbent assay (ELISA), and microfluidic chip using antibody-labeled streptavidin-biotin, fluorescent-labeled probes, giant magnetoresistive (GMR) sensors, probe-functionalized nanoparticles, magnetic nanoclusters (MNCs), and line probe assay (LiPA) [9,23]. Incorporating one of the platforms into LAMP detection system could potentially increase the sensitivity, as well as the cost per reaction.…”

“…LAMP utilizes a Bst polymerase for target amplification under a constant single temperature with a 30-to 60-min incubation [8]. The amplified product can be detected by various methods [9], but turbidity detection is the most promising means to be optimized toward both arms of accuracy and cost-effectiveness [10]. Although several HBV LAMP protocols have been described, limitations were still noted whenever applying to point of care.…”

Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection ofCryptosporidiumSpecies in Kenyan Children Presenting with Diarrhea

“…LAMP has been used in the diagnosis of protozoan parasitic pathogens such as Plasmodium falciparum [ 24 ], human infective Trypanosoma rhodesiense [ 21 ] and pathogens found in stool such as Ascaris lumbricoides [ 25 ] and Necator americanus [ 26 ]. Various methods have been used to detect LAMP products [ 27 ] in particular; the use of lateral flow dipstick (LFD) method offers high test specificity and visual inspection of results. This is because the detection probe targets a specific complementary sequence in the product and the results appear as presence or absence of a line on LFD stick [ 28 , 29 ].…”

Peroxidyme‐Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter

“…Chemical reactions that produce visible color changes have become indispensable to modern analytics and diagnostics, as evidenced by the widespread use of enzyme immunoassays, antibiotic‐resistant assays, nanoparticle‐aggregation assays, and many others. Typically, the signals produced by these assays are directly proportional to the analyte concentration .…”