MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method.

Fedor V Shirshikov,Yuri A Pekov,Konstantin A Miroshnikov

doi:10.7717/peerj.6801

Abstract

BackgroundAdvantages of loop-mediated isothermal amplification in molecular diagnostics allow to consider the method as a promising technology of nucleic acid detection in agriculture and medicine. A bioinformatics tool that provides rapid screening and selection of target nucleotide sequences with subsequent taxon-specific primer design toward polymorphic orthologous genes, not only unique or conserved common regions of genome, would contribute to the development of more specific and sensitive diagnostic assays. However, considering features of the original software for primer selection, also known as the PrimerExplorer (Eiken Chemical Co. LTD, Tokyo, Japan), the taxon-specific primer design using multiple sequence alignments of orthologs or even viral genomes with conservative architecture is still complicated.FindingsHere, MorphoCatcher is introduced as a fast and simple web plugin for PrimerExplorer with a clear interface. It enables an execution of multiple-alignment based search of taxon-specific mutations, visual screening and selection of target sequences, and easy-to-start specific primer design using the PrimerExplorer software. The combination of MorphoCatcher and PrimerExplorer allows to perform processing of the multiple alignments of orthologs for informative sliding-window plot analysis, which is used to identify the sequence regions with a high density of taxon-specific mutations and cover them by the primer ends for better specificity of amplification.ConclusionsWe hope that this new bioinformatics tool developed for target selection and taxon-specific primer design, called the MorphoCatcher, will gain more popularity of the loop-mediated isothermal amplification method for molecular diagnostics community. MorphoCatcher is a simple web plugin tool for the PrimerExplorer software which is freely available only for non-commercial and academic users at http://morphocatcher.ru.

Highlights

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification (Notomi et al, 2000), which was developed to eliminate disadvantages of polymerase chainHow to cite this article Shirshikov FV, Pekov YA, Miroshnikov KA. 2019
To explain main functions of the MorphoCatcher, we describe a protocol for designing taxon-specific primers using the most widely-known bioinformatics online tools, including the NCBI Nucleotide database, NCBI BLAST, Clustal Omega, and PrimerExplorer (Fig. 1)
The protocol contains several steps that help to find potential target sequence for diagnostic primers, keep the correct file formatting basing on well-known standards in bioinformatics, and obtain candidate primer sets for the LAMP experiments

Summary

Introduction

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification (Notomi et al, 2000), which was developed to eliminate disadvantages of polymerase chainHow to cite this article Shirshikov FV, Pekov YA, Miroshnikov KA. 2019. MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method. A bioinformatics tool that provides rapid screening and selection of target nucleotide sequences with subsequent taxon-specific primer design toward polymorphic orthologous genes, unique or conserved common regions of genome, would contribute to the development of more specific and sensitive diagnostic assays. MorphoCatcher is introduced as a fast and simple web plugin for PrimerExplorer with a clear interface It enables an execution of multiple-alignment based search of taxon-specific mutations, visual screening and selection of target sequences, and easy-to-start specific primer design using the PrimerExplorer software. We hope that this new bioinformatics tool developed for target selection and taxon-specific primer design, called the MorphoCatcher, will gain more popularity of the loop-mediated isothermal amplification method for molecular diagnostics community. MorphoCatcher is a simple web plugin tool for the PrimerExplorer software which is freely available only for non-commercial and academic users at http://morphocatcher.ru

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Discussion

Conclusion