Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

• Autor(a) principal: Venkatesan,Vasuki
• Data de Publicação: 2013
• Outros Autores: Hoti,Sugeerappa Laxmanappa, Kamaraj,Nagalakshmi, Ghosh,Somnath, Rajaram,Kaushik
• Tipo de documento: Relatório
• Idioma: eng
• Título da fonte: Memórias do Instituto Oswaldo Cruz
• Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000600804
• topic: asymmetric PCR; single-stranded DNA; electrochemical detection; Wuchereria bancrofti

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.