In-Silico Automated Allele-Specific Primer Design for Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
George Alexandrou ◽  
Jesus Rodriguez-Manzano ◽  
Kenny Malpartida-Cardenas ◽  
Pantelis Georgiou ◽  
Chris Toumazou ◽  
...  
Keyword(s):  
In Silico ◽  
Primer Design ◽  
Isothermal Amplification ◽  
Specific Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Allele Specific

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

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6801
Author(s):  
Fedor V. Shirshikov ◽  
Yuri A. Pekov ◽  
Konstantin A. Miroshnikov
Keyword(s):  
Molecular Diagnostics ◽  
Target Selection ◽  
Primer Design ◽  
Multiple Alignment ◽  
Isothermal Amplification ◽  
Specific Primer ◽  
Bioinformatics Tool ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Selection Of

Background Advantages 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. Findings Here, 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. Conclusions 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.

scholarly journals Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

2022 ◽  
Author(s):  
Rebecca Allsopp ◽  
Georgios Alexandrou ◽  
Christofer Toumazou ◽  
Simak Ali ◽  
Charles Coombes ◽  
...  
Keyword(s):  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Loop Formation ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Allele ◽  
Allele Specific ◽  
Specific Reactions ◽  
Circulating Tumour Dna

Abstract Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

scholarly journals A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac

2019 ◽  
Vol 20 (8) ◽  
pp. 1937 ◽  
Author(s):  
Miaomiao Sun ◽  
Hao Liu ◽  
Junbin Huang ◽  
Jinbo Peng ◽  
Fuhua Fei ◽  
...  
Keyword(s):  
Target Genes ◽  
Early Stage ◽  
Lamp Assay ◽  
Soft Rot ◽  
Isothermal Amplification ◽  
Comparative Genomic ◽  
Specific Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Accurate Detection ◽  
Specificity And Sensitivity

Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 104 CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.

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