Detection and discrimination of recombinant plasmid encoding hepatitis C virus core/E1 gene based on PNA and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for direct detection and discrimination of double-stranded plasmid (ds-Pl) without the need for denaturation of the target plasmid sample using a peptide nucleic acid (PNA) oligomer as the probe is described. This goal was achieved by modification of gold electrode with 6-mercapto-1-hexanol following monolayer self-assembly of cysteine conjugated 20-mer PNA oligomer probe, complementary to the HCV core/E1 region, which binds to ds-Pl and forms PNA/ds-Pl structure. The significant variation in differential pulse voltammetric response of methylene blue on the probe modified electrode upon contacting with complementary double-strand plasmid to form PNA/ds-Pl triplex structure is the principle of target plasmid detection. The results indicated that the reduction peak current was linear with the concentration of complementary strand in the range of 10–300 pg/μl with a detecion limit of 9.5 pg/μl.

► An electrochemical DNA biosensor for direct detection of target DNA in double–stranded plasmid.
► Direct detection of target DNA in ds-DNA form using PNA oligomer as probe.
► Simplification of assay protocol via elimination of ds-DNA denaturation step into ss-DNA.
► Detection and determination of target plasmid with desirable detection limit.