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

doi:10.1021/acssensors.1c01710

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.

Highlights

We report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides
To use labeled dNTPs rather than ddNTPs, we explored the possibility of solid-phase primer elongation for the detection of single-nucleotide polymorphisms (SNPs)
The electrochemical detection of solid-phase primer elongation from gold electrode surfaces using ferrocene-labeled oligonucleotides has been exploited for the detection of singlenucleotide mutations

Summary

■ RESULTS AND DISCUSSION

We selected a 128 mer oligonucleotide that includes an 81 nt RIF-resistance determining region (RRDR) of the beta subunit of the RNA polymerase (rpoB) gene plus primer regions as a model target. The master mix contains the mixture of dNTPs (concentrations of the labeled and unlabeled dNTPs are detailed in Table S2), one primer, which is fully complementary with the target, the ssDNA target, and the Klenow (exo-) DNA polymerase, as described in Section 3 of the Supporting Information and Figure S4. 50%, correlated to the results obtained using solution-phase primer elongation This may be attributable to a better substrate specificity of the Klenow (exo-) DNA polymerase for dAFcTP as compared to dGFcTP and dUFcTP, as well as the possible steric hindrance between consecutive ferrocenelabeled bases at homopolymeric regions of the amplicon, which could inhibit the elongation at higher percentages of dNFcTP, as was previously observed for other bulky electroactive labels.[54]. The base detected at the SNP site (positive primer A, SNP T) correlated with the results obtained by sequencing

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES

Detection of Mitochondrial Single Nucleotide Polymorphisms