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.
Highlights
Lymphatic filariasis (LF), one of the most important neglected tropical diseases, continues to batter the social and economic wellbeing of people in underdeveloped/ developing nations
The utility of the purified single-stranded DNA (ssDNA) generated in detecting W. bancrofti parasite DNA was verified by electrochemical detection using a prototypic electrochemical cell (E-cell)we developed and a cyclic voltammeter (CV)
In a search for new tools and strategies for monitoring and evaluating filariasis for elimination campaigns, a nanotechnology-based approach was attempted for the sequence-specific electrochemical detection of W. bancrofti-specific polymerase chain reaction (PCR) products. ssDNA is a prerequisite for the electrochemical sensor-based detection
Summary
Lymphatic filariasis (LF), one of the most important neglected tropical diseases, continues to batter the social and economic wellbeing of people in underdeveloped/ developing nations. LF is caused by the lymphatic system-dwelling nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori and is transmitted by vector mosquitoes. It has been targeted for an elimination programme that is largely based on repeated annual cycles of mass drug administration (MDA) using an antifilarial drug against endemic populations (Ottesan 2000). We designed an electrochemical DNA biosensor consisting of an electrode surface modified with an oligonucleotide probe complimentary to a target DNA analyte. An asymmetric PCR technique was optimised to generate an excess of ssDNA for use in electrochemical detection. This enabled us to achieve the sensitivity and reproducibility of electrochemical detection and the results are presented here
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