Background Despite the recent development of different detection methods, better diagnostic tools are required for quick and reliable detection of pathogens. Use of biosensors for detection of pathogens is gradually gaining momentum. However, PCR amplification of DNA target is still necessary for application on biosensor for accurate detection of the pathogen. An in-house PCR using self-designed primers targeting the opa gene (GenBank accession no. PUID 9716120 SNUM 2706 Ng_opa) was performed. The generated amplicons were used to evaluate the DNA biosensor utilising a 19-mer oligonucleotide sequence (GenBank PUID SNUM: 9716119 2705 Ng_opa) as probe.
Methods In-house PCR was standardised and an amplified product (amplicons) of 188 bps were obtained for positive samples. Fabrication of bioelectrode was performed by immobilising the activated probe onto pre-treated screen-printed gold electrodes. The fabricated nucleic acid functionalised gold electrode was characterised using, SEM, CV, DPV techniques. The presence of target DNA was detected electrochemically by monitoring the redox peak of methylene blue indicator. Standardisation of working conditions was done using complementary, non-complementary, one base mismatch DNA, and amplicons from standard strain of N. gonorrhoeae (ATCC 49226) & 16 clinical isolates. In addition, DPV measurements of hybridised bioelectrode with amplicons of 26 clinical samples of which 10 were culture positive, was done. A cut-off value for positives was determined by using the software STATA (version 9).
Results The analytical sensitivity of PCR was 10–17 M of DNA and the bioelectrode could detect up to 1.0×10–20 M of the DNA amplicons. An 11.49% decrease in signal intensity was taken as the cut-off. Samples giving an equivalent or more decrease than this value were considered as positives.
Conclusions The coupling of electrochemical detection with PCR amplification showed the advantage of higher sensitivity and increased specificity for detection of N gonorrhoeae. This may prove to be particularly valuable for the identification of asymptomatic infections and could greatly improve gonorrhoea control.
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