Development and application of a rapid semiquantitative immunochromatographic test strip to detect white spot syndrome virus

摘要

White spot syndrome virus (WSSV) is one of the most serious viral pathogens causing major economic losses to the shrimp farming industry worldwide. Rapid diagnostic test is critical for disease prevention and control. Previously we have developed a rapid immunochromatographic test strip for WSSV detection, but it is limited to the qualitative testing. In this study, a semiquantitative immunochromatographic test strip (SIT-strip) was developed for WSSV detection by observing the number of positive test lines to determine the viral load levels. To achieve semiquantitative analytical ability, three test lines were designed on NC membrane to form the test zone, on which different concentration of anti-WSSV monoclonal antibody (MAb) 4G9 in a decreasing sequence from test line (TL) I to TL-III was dispensed, instead of the traditional application of a single test line. The anti-WSSV MAb 2E6 served as colloidal gold conjugate and goat anti-mouse IgG antibody on control line was used to validate the test performance. The threshold concentrations for TL-I, -II and -III were determined to be 783 ng/ml, 3.13 mu g/ml and 6.26 mu g/ml of WSSV, respectively, and the visual detection limit of the SIT-strip was 783 ng/ml. The developed SIT-strip was used to detect WSSV at different time points post injection of Procambarus clarkia, the results indicated that one red band was present on TL-I at 12 h post infection (hpi) in the gills, two bands in muscle and three bands in gills and hemolymph were produced at 24 hpi although there was no death at that time, and thereafter three bands were visible in all tested tissues and dead individuals. The mortality was low from 36 to 48 hpi and increased rapidly during 48-72 hpi. Meanwhile, WSSV copy numbers were detected by real time qPCR for severity grading of infection. These results revealed that this SIT-strip could provide confirmation of viral presence and concentration levels prior to mortality, which was early enough to achieve the goal of monitoring for WSSV infection loads predictive of impending disease.