title: Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection.
abstract: Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.
authors.affiliation: Vector Control Research Centre, Indira Nagar Medical Complex, Pondicherry, India.
authors.forename: Vasuki
authors.initials: V
authors.lastname: Venkatesan
authors.forename: Sugeerappa Laxmanappa
authors.initials: SL
authors.lastname: Hoti
authors.forename: Nagalakshmi
authors.initials: N
authors.lastname: Kamaraj
authors.forename: Somnath
authors.initials: S
authors.lastname: Ghosh
authors.forename: Kaushik
authors.initials: K
authors.lastname: Rajaram
mesh_terms: D000818:Animals; D015342:DNA Probes; D004277:DNA, Single-Stranded; D055664:Electrochemical Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D014958:Wuchereria bancrofti / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes
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