Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-034-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 15, 2022
End Date: Aug 14, 2024
This collaborative agreement aims to develop a nanoparticle-based DNA biosensor to rapidly detect African Swine Fever Virus (ASFV) vaccine strains and discriminate between infected and vaccinated animals (DIVA). 1. Design oligonucleotide probes to detect ASFV conserved genes and validate their sensitivity and specificity; 2. Design oligonucleotide probes to detect the absence of deleted genes and validate their sensitivity and specificity; 3. Design oligonucleotide probes to detect the presence of the vaccine biomarker gene and validate their sensitivity and specificity; and 4. Develop a preliminary proof-of-concept multiplex assay to simultaneously verify the presence of p72 and mCherry genes and the absence of I177L gene.
Un-amplified genomic DNA, specific oligonucleotide probes, and gold nanoparticles (GNPs) are mixed in a tube. The GNP and oligo probes are conjugated through the amine-thiol interaction, and the oligo probes hybridize with the target DNA, forming a stable GNP-probe-DNA complex. The solution is protonated by adding an acid, and the color change is observed: red for the presence of the target DNA due to the formation of the GNP-probe-DNA complex; blue if the target DNA is absent. The color is observable to the naked eye and can be captured by the smartphone App to be developed. The color is converted into an RGB scale, which is then used to report the presence or absence of the target gene digitally. Using specific probes for specific genes in ASFV will allow the fast differentiation between circulating ASFV strains and vaccines.