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.
Keywords: Ag/AgCl; Aphanomyces invadans; AuNPs; BSA; CP; DPASV; EUS; Epizootic ulcerative syndrome; Genosensor; Gold nanoparticles; ITS; Multi-layer latex particles; PAA; PB; PCR; PCR product; PSA; PSS; Pt; RP; SPE; TEM; bovine serum albumin; capture probe; differential pulse anodic stripping voltammetry; epizootic ulcerative syndrome; gold nanoparticles; internal transcribed spacer regions; phosphate buffer; platinum; poly(allylamine) hydrochloride; poly(sodium 4-styrene) sulfonate; polymerase chain reaction; polystyrene-co-acrylic acid; reporter probe; screen printed electrode; silver–silver chloride; transmission electron microscope.
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