Strategies for rapid and reagent-less electrochemical detection of RPA products

Identificador:  TDX:2893

Handle: https://hdl.handle.net/20.500.11797/TDX2893

Autors:  Sallam Mohammed Ahmed, Al-Madhagi

Resum:
Nowadays, there is a need to develop a rapid, simple, inexpensive and reliable DNA testing system for diagnosis in different fields such as genetic diseases, pathogens detection, forensics, and personalised medicine. Conventional methods for the detection of specific DNA sequences are based on direct sequencing or hybridisation assays, being this last one approach, the most widely used in genosensors. It consists on single-stranded DNA (ssDNA) tethered probes on a transducer surface, which recognises its complementary sequence (target) with high affinity and specificity. One of the limitations of these DNA sensors to apply them for a portable molecular diagnostics devices is the multi-step procedures needed, since post-amplification treatment is necessary for its detection by generating ssDNA or adding a hapten labelling. In this work, the isothermal amplification and modified tailed primers to simplify the steps required for the electrochemical DNA detection are combined. Modified tailed primers are based on a single stranded oligonucleotide sequence linked to a carbon spacer, which effectively blocks elongation, prior to the primer sequence. Thus, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. One of the tails was used to hybridise to a surface immobilised probe and the other to an enzyme or gold nanoparticles labelled reporter probe. Using these modified primers allowed us to detect DNA electrochemically without any need for post-amplification sample treatment decreasing the assay time and presenting an approach that can facilely find application at the point of need.