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

0550081 - ÚOCHB 2022 RIV US eng J - Článek v odborném periodiku
Ortiz, M. - Jauset-Rubio, M. - Skouridou, V. - Machado, D. - Viveiros, M. - Clark, T. G. - Simonova, Anna - Kodr, David - Hocek, Michal - O'Sullivan, C. K.
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. Roč. 6, č. 12 (2021), s. 4398-4407. ISSN 2379-3694. E-ISSN 2379-3694
Grant CEP: GA ČR(CZ) GX20-00885X
Institucionální podpora: RVO:61388963
Klíčová slova: single-point mutation * single-nucleotide polymorphism (SNP) * ferrocene-labeled nucleotides * nucleoside triphosphates * solid-phase primer elongation * Klenow (exo-) DNA polymerase
Obor OECD: Organic chemistry
Impakt faktor: 9.618, rok: 2021
Způsob publikování: Open access
https://doi.org/10.1021/acssensors.1c01710

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.
Trvalý link: http://hdl.handle.net/11104/0325929