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Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers.
Malpartida-Cardenas, Kenny; Rodriguez-Manzano, Jesus; Yu, Ling-Shan; Delves, Michael J; Nguon, Chea; Chotivanich, Kesinee; Baum, Jake; Georgiou, Pantelis.
Afiliación
  • Malpartida-Cardenas K; Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering , Imperial College London , London , SW7 2AZ , United Kingdom.
  • Rodriguez-Manzano J; Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering , Imperial College London , London , SW7 2AZ , United Kingdom.
  • Yu LS; Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering , Imperial College London , London , SW7 2AZ , United Kingdom.
  • Delves MJ; Department of Life Sciences, Imperial College London , South Kensington Campus , SW7 2AZ , London , United Kingdom.
  • Nguon C; National Centre for Parasitology , Entomology and Malaria Control , Phnom Penh 12302 , Cambodia.
  • Chotivanich K; Department of Clinical Tropical Medicine, Faculty of Tropical Medicine , Mahidol University , Bangkok 10400 , Thailand.
  • Baum J; Department of Life Sciences, Imperial College London , South Kensington Campus , SW7 2AZ , London , United Kingdom.
  • Georgiou P; Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering , Imperial College London , London , SW7 2AZ , United Kingdom.
Anal Chem ; 90(20): 11972-11980, 2018 10 16.
Article en En | MEDLINE | ID: mdl-30226760
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plasmodium falciparum / Termodinámica / Polimorfismo de Nucleótido Simple / Técnicas de Amplificación de Ácido Nucleico / Secuencia Kelch Tipo de estudio: Guideline Límite: Humans Idioma: En Revista: Anal Chem Año: 2018 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plasmodium falciparum / Termodinámica / Polimorfismo de Nucleótido Simple / Técnicas de Amplificación de Ácido Nucleico / Secuencia Kelch Tipo de estudio: Guideline Límite: Humans Idioma: En Revista: Anal Chem Año: 2018 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Estados Unidos