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Updated: Oct 11, 2025
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of
Mayreli Ortiz1, Miriam Jauset-Rubio1, David Kodr2
1Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
Insights
This study developed a novel electrochemical platform to detect hypertrophic cardiomyopathy (HCM)-associated single nucleotide polymorphisms (SNPs) in MYH7 gene from fingerprick blood. The assay accurately identifies SNPs using minimal DNA, offering a promising diagnostic tool for HCM.
Area of Science:
- Biomedical Engineering
- Molecular Diagnostics
- Cardiovascular Genetics
Background:
- Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death in young individuals.
- An estimated 1 in 500 people are affected by HCM.
- Genetic mutations, particularly in the MYH7 gene, are strongly associated with HCM.
Purpose of the Study:
- To develop an electrochemical platform for detecting HCM-associated single nucleotide polymorphisms (SNPs).
- To enable detection using small-volume fingerprick blood samples.
- To provide a rapid and accurate diagnostic method for HCM genetic screening.
Main Methods:
- Development of an electrochemical array with gold electrodes.
- Immobilization of thiolated reverse primers with variable bases.
- Utilizing isothermal solid-phase primer elongation with recombinase polymerase amplification (RPA).
- Incorporation of ferrocene-labelled nucleoside triphosphates for signal detection.
Main Results:
- Successful discrimination of HCM-associated SNPs in the MYH7 gene.
- Assay validated on fingerprick blood samples from eight individuals.
- Results showed strong correlation with next-generation sequencing data.
- Demonstrated robust SNP detection at low femtomolar DNA concentrations (approx. 8 × 10^5 copies).
Conclusions:
- The developed electrochemical platform offers a sensitive and specific method for HCM SNP detection.
- The assay's ability to use fingerprick blood samples enhances accessibility for genetic screening.
- This technology holds potential for early diagnosis and management of hypertrophic cardiomyopathy.
Abstract:
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.
