Design and establish a rapid diagnostic kit for Viral Nervous Necrosis (VNN) disease using immunochromatography method

Abstract

Background: Our investigation has approved that Viral Nervous Necrosis was the main causative agent for the outbreak in Mullet fish in the Caspian Sea. The disease was first reported in wild golden grey mullet (Liza auratus) in 2004. Since betanodaviruses are completely resistant to environmental conditions, it is possible to move easily via commercial activities. Also, the virus can introduce horizontally through contaminated water. So, the rapid diagnosis could be very useful in control, prevention and eradication programs. Also, in Monitoring & Surveillance, fast detection of the unknown outbreak could be vital and decrease their economic impacts. Methods and materials: For the preparation of antibody-gold particle conjugates, the concentration of Abs and pH of colloidal gold should be adjusted. A different volume of 1% sodium citrate was added to obtain the desired gold nanoparticles size in 30 nm. It was conjugated with an appropriate concentration Ab and in specific pH, and then this conjugation was blocked with serum albumin bovine. Afterwards, both various volumes of antibody-gold particle conjugates were coated on the conjugate pad and different concentration of pAb are used in a test line. To assure that the test strip will work correctly, the flow must also reach the control line, which has goat anti-mouse IgG (5 mg/ml) embedded in it. Both test and control lines were spotted on the membrane by using Helena applicator. Finally, the components of the strip were assembled and then the brain specimens of suspicious grey mullets and different dilution of the virus were evaluated. Results: With using of 1 μg/μl of pAbs on the test line and 4 μl per strip of gold- antibody conjugates on the conjugate pad, the designed strip was able to detect 103 TCID50/ml concentration of VNN. Then 50 brain samples that produced positive test results by PCR. The supernatant solution from brain homogenate of VNN-infected Liza aurata was confirmed negative for VNN by subsequent PCR an IHC testing, were evaluated using immunochromatography strip. It was recognized that the sensitivity and specificity of this kit are 64 and 100%, respectively. Conclusion: The accuracy of the immunochromatography kit was identical to both real-time PCR and IHC.