Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata)

Abstract

A lateral flow immunochromatography strip test, based on antibody–gold nanoparticles specific for nervous necrosis virus (NNV), was developed for rapid, on‐site detection of the virus in fish stocks. A monoclonal antibody against NNV was conjugated with colloidal gold as the detector antibody. A rabbit anti‐NNV polyclonal antibody and goat anti‐mouse IgG antibody were blotted onto the nitrocellulose membrane as the capture antibodies on the test line and control line, respectively. The reaction could be seen by the eye within 15 min and did not cross‐react with the other viruses tested. The detection limit of the strip was approximately 103 TCID50/ml and had good stability after storage at 4°C for 8 months. When brains of 70 naturally infected golden grey mullet, Chelon aurata, were tested with the strip test, the diagnostic specificity and sensitivity of the test compared to real‐time RT‐PCR were 100% and 74%, respectively. Therefore, the one‐step test strip developed here had high specificity, reproducibility, and stability. This, together with its simplicity to use and rapid detection, without the requirement of sophisticated equipment or specialized skills, makes the strip suitable for pond‐side detection of NNV in farmed fish.

Trace Determination of Grouper Nervous Necrosis Virus in Contaminated Larvae and Pond Water Samples Using Label-Free Fiber Optic Nanoplasmonic Biosensor

“…Although no fish died during the first eight days of the culturing process, NNV coat protein concentration was found to be 1.7 × 10 −7 g/mL on the eighth day. This concentration level was one order of magnitude lower than the reported LOD using the ELISA method [ 7 ]. The detected concentration of NNV coat proteins gradually increased after the eighth day.…”

“…While conventional enzyme-linked immunosorbent assay (ELISA) methods provide adequate limit of detection (LOD) for determining NNV coat proteins in brain homogenate samples of infected fish containing more than 3 µg/mL coat proteins [ 7 ], this LOD is not capable of detecting NNV in slightly contaminated aquaculture water at the viral titer level of 10 3.5 TCID 50 /mL, which is estimated to contain about 0.1 µg/mL crude proteins [ 7 ] and is equivalent to 10 6 NNV copies/mL [ 8 ]. Besides, performing ELISA on site is difficult, limiting its applications in resource-limited areas.…”