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Allele-specific isothermal amplification method using novel unmodified self-stabilizing competitive primers

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Title: Allele-specific isothermal amplification method using novel unmodified self-stabilizing competitive primers
Authors: Malpartida-Cardenas, K
Rodriguez-Manzano, J
Yu, L-S
Delves, M
Nguon, C
Chotivanich, K
Baum, J
Georgiou, P
Item Type: Journal Article
Abstract: Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance.
Issue Date: 16-Oct-2018
Date of Acceptance: 18-Sep-2018
URI: http://hdl.handle.net/10044/1/64956
DOI: https://dx.doi.org/10.1021/acs.analchem.8b02416
ISSN: 0003-2700
Publisher: American Chemical Society
Start Page: 11972
End Page: 11980
Journal / Book Title: Analytical Chemistry
Volume: 90
Issue: 20
Copyright Statement: © 2018 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Sponsor/Funder: Wellcome Trust
Wellcome Trust
Funder's Grant Number: 100993/Z/13/Z
100993/Z/13/Z
Keywords: Science & Technology
Physical Sciences
Chemistry, Analytical
Chemistry
POLYMERASE-CHAIN-REACTION
TEMPLATE MISMATCHES
HIGH-THROUGHPUT
HIGH-ACCURACY
DNA
THERMODYNAMICS
TEMPERATURE
PCR
0301 Analytical Chemistry
0904 Chemical Engineering
0399 Other Chemical Sciences
Analytical Chemistry
Publication Status: Published
Online Publication Date: 2018-09-18
Appears in Collections:Electrical and Electronic Engineering
Faculty of Natural Sciences



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