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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 × 10<sup>1</sup> 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.

Original publication





Analytical chemistry

Publication Date





11972 - 11980


Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering , Imperial College London , London , SW7 2AZ , United Kingdom.


Humans, Plasmodium falciparum, DNA Primers, Nucleic Acid Amplification Techniques, Polymorphism, Single Nucleotide, Alleles, Thermodynamics, Kelch Repeat