A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.
Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.