Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides

Mayreli Ortiz, Miriam Jauset-Rubio, Vasso Skouridou, Diana Machado, Miguel Viveiros, Taane G. Clark, Anna Simonova, David Kodr, Michal Hocek, Ciara K. O'Sullivan

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5′-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2′-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

Original languageEnglish
Pages (from-to)4398–4407
Number of pages10
JournalACS Sensors
Volume6
Issue number12
Early online date19 Nov 2021
DOIs
Publication statusPublished - 24 Dec 2021

Keywords

  • ferrocene-labeled nucleotides
  • Klenow (exo-) DNA polymerase
  • nucleoside triphosphates
  • single-nucleotide polymorphism (SNP)
  • single-point mutation
  • solid-phase primer elongation

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