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Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides
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Resumo(s)
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.
Descrição
Funding Information: This project received partial funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 767325, the Czech Science Foundation (20-00885X to M.H.), and the Fundação para a Ciência e a Tecnologia, Portugal (Grant - UID/Multi/04413/2020). The authors thank Fraunhofer ICT-IMM for the provision of gold electrode arrays. The Ph.D. scholarship of D. Kodr from the Department of Chemistry of Natural Compounds of the University of Chemistry and Technology Prague is also acknowledged. Publisher Copyright: © 2021 The Authors. Published by American Chemical Society.
Palavras-chave
ferrocene-labeled nucleotides Klenow (exo-) DNA polymerase nucleoside triphosphates single-nucleotide polymorphism (SNP) single-point mutation solid-phase primer elongation Bioengineering Instrumentation Process Chemistry and Technology Fluid Flow and Transfer Processes Infectious Diseases SDG 3 - Good Health and Well-being SDG 9 - Industry, Innovation, and Infrastructure SDG 10 - Reduced Inequalities SDG 12 - Responsible Consumption and Production SDG 17 - Partnerships for the Goals