Articles producció científica> Enginyeria Química

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

  • Identification data

    Identifier: imarina:9242276
    Authors:
    Ortiz, MayreliJauset-Rubio, MiriamSkouridou, VassoMachado, DianaViveiros, MiguelClark, Taane GSimonova, AnnaKodr, DavidHocek, MichalO'Sullivan, Ciara K
    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. © 2021 The Authors. Published by American Chemical Society.
  • Others:

    Author, as appears in the article.: Ortiz, Mayreli; Jauset-Rubio, Miriam; Skouridou, Vasso; Machado, Diana; Viveiros, Miguel; Clark, Taane G; Simonova, Anna; Kodr, David; Hocek, Michal; O'Sullivan, Ciara K
    Department: Enginyeria Química
    URV's Author/s: Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Skouridou, Vasoula
    Keywords: Solid-phase primer elongation Solid-phase Solid phasis Single-point mutation Single-nucleotide polymorphism (snp) Single-nucleotide polymorphism Single nucleotide polymorphisms Rifampin Polymorphism, single nucleotide Polymorphism Oxidation-reduction Organometallics Oligonucleotides Nucleoside triphosphates Mycobacterium tuberculosis Metallocenes Klenow (exo-) dna polymerase Iron compounds Ferrocene-labeled nucleotides Ferrocene-labeled nucleotide Ferrocene labelled Elongation Electrodes Dna polymerase Dna Cost effectiveness Chemical detection
    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. © 2021 The Authors. Published by American Chemical Society.
    Thematic Areas: Process chemistry and technology Nanoscience & nanotechnology Instrumentation Fluid flow and transfer processes Engenharias iii Chemistry, multidisciplinary Chemistry, analytical Bioengineering
    licence for use: https://creativecommons.org/licenses/by/3.0/es/
    Author's mail: vasoula.skouridou@urv.cat mayreli.ortiz@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat
    Author identifier: 0000-0002-9712-5429 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055
    Record's date: 2024-10-12
    Papper version: info:eu-repo/semantics/publishedVersion
    Link to the original source: https://pubs.acs.org/doi/10.1021/acssensors.1c01710
    Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
    Papper original source: Acs Sensors. 6 (12): 4398-4407
    APA: Ortiz, Mayreli; Jauset-Rubio, Miriam; Skouridou, Vasso; Machado, Diana; Viveiros, Miguel; Clark, Taane G; Simonova, Anna; Kodr, David; Hocek, Michal; (2021). Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides. Acs Sensors, 6(12), 4398-4407. DOI: 10.1021/acssensors.1c01710
    Article's DOI: 10.1021/acssensors.1c01710
    Entity: Universitat Rovira i Virgili
    Journal publication year: 2021
    Publication Type: Journal Publications
  • Keywords:

    Bioengineering,Chemistry, Analytical,Chemistry, Multidisciplinary,Fluid Flow and Transfer Processes,Instrumentation,Nanoscience & Nanotechnology,Process Chemistry and Technology
    Solid-phase primer elongation
    Solid-phase
    Solid phasis
    Single-point mutation
    Single-nucleotide polymorphism (snp)
    Single-nucleotide polymorphism
    Single nucleotide polymorphisms
    Rifampin
    Polymorphism, single nucleotide
    Polymorphism
    Oxidation-reduction
    Organometallics
    Oligonucleotides
    Nucleoside triphosphates
    Mycobacterium tuberculosis
    Metallocenes
    Klenow (exo-) dna polymerase
    Iron compounds
    Ferrocene-labeled nucleotides
    Ferrocene-labeled nucleotide
    Ferrocene labelled
    Elongation
    Electrodes
    Dna polymerase
    Dna
    Cost effectiveness
    Chemical detection
    Process chemistry and technology
    Nanoscience & nanotechnology
    Instrumentation
    Fluid flow and transfer processes
    Engenharias iii
    Chemistry, multidisciplinary
    Chemistry, analytical
    Bioengineering
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