Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of single nucleotide polymorphisms

0550788 - ÚOCHB 2023 RIV NL eng J - Journal Article
Ortiz, M. - Jauset-Rubio, M. - Kodr, David - Simonova, Anna - Hocek, Michal - O'Sullivan, C. K.
Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of single nucleotide polymorphisms.
Biosensors and Bioelectronics. Roč. 198, February (2022), č. článku 113825. ISSN 0956-5663. E-ISSN 1873-4235
R&D Projects: GA ČR(CZ) GX20-00885X
Institutional support: RVO:61388963
Keywords : fingerprick * solid phase recombinase polymerase amplification * SNP * Ferrocene-dNTPs * biosensors * electrochemical detection
OECD category: Organic chemistry
Impact factor: 12.6, year: 2022
Method of publishing: Open access
https://doi.org/10.1016/j.bios.2021.113825

Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3’ end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 μL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.
Permanent Link: http://hdl.handle.net/11104/0326095