Development Of A Semiquantitative Immunochromatographic Test Strip(SIT-strip)to Detect White Spot Syndrome Virus

White spot syndrome virus (WSSV) is themost severe viral pathogen to the shrimp culture industry worldwide. There are no effective methods to cure this desease right now, so early detection of WSSV in infected shrimp is very important that effective measures can be taken immediately. Various methods are available for the quantitative detection of WSSV, such as polymerase chain reaction (PCR), real-time quantitative PCR, double antibody sandwich ELISA and so on. However, their performances are restricted to laboratory mainly due to the high-cost, Time-consuming and requirement of skilled sophisticated equipments. In response to these circumstances, we development a semiquantitative immunochromatographic test strip(SIT-strip)to detect White Spot Syndrome Virus of shrimp, and the test process can be completed within 10 min without incubation or any equipment which is suitable for rapid detection of semi-quantitative of WSSV for shrimp, crabs and other crustaceans.Five strains of hybridomas (2E6、1G12、3B7、4G9、5D2) secreting monoclonal antibodies (McAbs) against WSSV were cultured, and then ascitic fluids were obtained and purified by caprylic acid ammonium sulphate method. Finally, McAb 2E6 was selected as the capture antibody labeled with colloidal gold for the GICA based on the principle of double antibody sandwich. Monodispersed colloidal gold with a mean diameter of approximately 20 nm was synthesized by sodium citrate reduction. It was proved that colloidal gold was homogeneous, without fragments and agglutination characterized by visual observation.Different concentrations of purified McAb 4G9 and goat anti-mouse IgG were coated on the nitrocellulose membrane as and the test linel, the test line2, the test line2, and the control line. Anti-WSSV MAbs mixture of 2A3,3B7,4G9 and 5D2 in a volume ratio 2:4:2:1 served as capture antibodies, which immobilized on Whatman AE99 nitrocellulose membrane (NCM).The dectection limit of the ICT was 783ng/ml. In the process of diagnosis, the WSSV antigen binds the colloidal gold-antibody conjugation on the test strip and moves along the membrane. Then it binds the capture antibody at the test line on nitrocellulose membrane displaying a visible red line. If the test linel,2,3 show a red line mean that concentrations of WSSV is greater than 6.26μg/ml; If only the test line1,2, show a red line mean that concentrations of WSSV is between 3.13μg/ml and 6.26 μg/ml; If only the test linel show a red line mean that concentrations of WSSV is between783ng/ml and 3.13 u g/ml, If only all the test line not show a red line mean that concentrations of WSSV is less than 783ng/ml or free of WSSV.The temporal and spatial dynamic changes of WSSV in Procambarus clarkii infected with WSSV were studied through the quantitative real-time PCR method and SIT-strip. Procambarus clarkii were intramuscularly injected with WSSV, The gill, hemolymph,heart, muscle,pleopods and hemopoietic tissue selected at 0,3,6,12,24, 36,39,48,60 and 72 hours post-injection. DNA was extracted from the samples and the viral load was studied through the quantitative real-time PCR method. WSSV was detected in pleopod, gill and hemolymph at 3 hours post-injection, and WSSV was detected in all sampling tissues at 12 hours post-injection, and the viral load was low; The viral load in the pleopod was increased logarithmically in the next 48 hours, and a low mortality rate was observed; And thereafter the WSSV propagation went into the plateau phage and massive mortality happened. Our data indicated that there was a positive correlation between the viral load and Cumulative mortality of Procambarus clarkii, and the dynamic changes of the amount of WSSV in pleopods can well represent the multiplication rule of WSSV in other sampling tissues so pleopods can be used as the reliable detection tissue not only in laboratory experiments but also in field surveys. The progress rate of WSSV in different tissues during the infection was different. According to the experimental results we speculate that the virus was first copied in the hemolymph, and then WSSV infected other organizations with hemolymph circulation. The viral load of gill, hemolymph and muscle was studied by SIT-strip, and the result of SIT-strip was consistent with real-time quantitative PCR at rate of 100%. So SIT-strip was a rapid, simple, convenient detection method, and it can be done pond side just using a test stripe kit.