Allele-specific isothermal amplification method using novel unmodified self-stabilizing competitive primers
File(s)
Published version
Author(s)
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.
Date Issued
2018-10-16
Date Acceptance
2018-09-18
Citation
Analytical Chemistry, 2018, 90 (20), pp.11972-11980
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.
License URL
Sponsor
Wellcome Trust
Wellcome Trust
Grant Number
100993/Z/13/Z
100993/Z/13/Z
Subjects
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
Date Publish Online
2018-09-18