Single-color multiplexing by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification

Abstract

We develop a single-color multiplexing strategy to identify multiple amplicons without the involvement of multicolor labels and parallelized multiplexing.

A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR

“…We speculate that the difference is because LAMP generates a large amount of DNA amplicons compared to PCR. Like in PCR, the melting profile of LAMP products is primarily dependent on GC content, sequence and length of the amplicons [ 35 ]. Gel electrophoresis can confirm the formation of a specific product in PCR with a single band of a given size.…”

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis

“…Here, we developed a multiplexing technique using fluorescent intercalating dye and melting curves analysis. This approach has been widely applied in PCR and has also been used for LAMP in two works [ 31 , 39 ]. Thus, analysis using standard real-time thermocyclers is available, without relatively expensive fluorescently labeled oligonucleotides or other specific equipment.…”

“…The concatemeric nature of LAMP products, consisting of repetitive amplified DNA fragments, hinders the separation of products without additional procedures. LAMP multiplexing can be performed using endonuclease digestion followed by gel-electrophoresis [ 26 , 27 ], modified oligonucleotides [ 28 , 29 , 30 ] or the melting of amplification products in the presence of intercalating dyes [ 31 , 32 ]. In the latter case, the products are differentiated based on their characteristic melting temperatures.…”

Naked‐eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens

“…One of its major drawbacks relies on the primer design, as very little software is available (LAMP designer and Primer explorer are the most common), and the difficulty for multiplex assay development. Some studies have been published elucidating different strategies for multiplexing; these include the usage of restriction enzymes to differentiate targets (Iseki et al., 2007), real‐time fluorescence detection followed by melt curve analysis (D'Agostino et al., 2015; Dong et al., 2019), and different types of fluorescent probes (Ball et al., 2016; Kubota & Jenkins, 2015; Tanner et al., 2012).…”