Publication: Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers
Issued Date
2018-10-16
Resource Type
ISSN
15206882
00032700
00032700
Other identifier(s)
2-s2.0-85054420438
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Mahidol University
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SCOPUS
Bibliographic Citation
Analytical Chemistry. Vol.90, No.20 (2018), 11972-11980
Suggested Citation
Kenny Malpartida-Cardenas, Jesus Rodriguez-Manzano, Ling Shan Yu, Michael J. Delves, Chea Nguon, Kesinee Chotivanich, Jake Baum, Pantelis Georgiou Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers. Analytical Chemistry. Vol.90, No.20 (2018), 11972-11980. doi:10.1021/acs.analchem.8b02416 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/45470
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Title
Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers
Abstract
Copyright © 2018 American Chemical Society. 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.