Development Of A Colloidal Gold-based Immunochromatographic Lateral Flow Assay For The Detection Of Shewanella Smarisflavi

Apostichopus japonicus is rich in mucopolysaccharides, proteins, amino acids, seacucumber saponins and other nutrients. Farming scale of Apostichopus japonicus grew rapidlyin our country because of its high economic value and nutritional value. But Apostichopusjaponicus disease problems have become increasingly prominent due to the immatureaquaculture pattern and the irregular operation during the production process. Skin ulcerationsyndrome caused by bacterial infection is the most serious disease and it’s very common inApostichopus japonicus aquaculture. This disease has pathogen diversity and regionaldifference. Shewanella smarisflavi is the important pathogen which could lead toApostichopus japonicus skin ulceration syndrome in liaoning, causing massive death ofApostichopus japonicus and resulting in huge economic loss during culture process. This kindof bacteria seriously threaten the development of sea cucumber aquaculture. The rapid andaccurate diagnosis is needed specific to disease in aquaculture animal. Usually diseaseoutbreak caused by pathogenic bacteria has a incubation period. During this period, symptomsare not obvious to see which resulted in disease diagnosis delays. Then we lose the best timefor treatment of the disease. Currently methods for detecting Shewanella smarisflavi includingphysiological and biochemical method, molecular biological method and ELISA needprofessional person. The operations are time consuming which cannot achieve at thegrassroots level. In order to achieve early diagnosis of the disease, a fast, simple and accuratemethod for pathogen detection is required.GICA is a rapid immune analytical technique marked by colloidal gold. The test stripestablished by this method is a neotype vitro assay tool. This method is simple and rapid, thereaction is amplified and the result can be seen directly. All the advantages can satisfy therequirement of field test to detect Shewanella smarisflavi. This method doesn’t needprofessional person which can prevent and control the disease effectively. In this study, weprepared monoclonal antibody and polyclonal antibody against Shewanella smarisflavi. TheGICA technology use double-antibody sandwich ELISA to detect Shewanella smarisflavi. Weoptimized index such as colloidal gold-labeled monoclonal antibody and antibody coating.We determine the best condition of this method and develop the colloidal goldimmunochromatographic strip successfully. The test strip developed to detect Shewanella smarisflavi can obtain the result in5to10minutes. The sensitivity of this method can limit to105CFU/mL, the best detectionconcentration is5×105~5×106CFU/mL. We use the strip testing two concentration,5×105CFU/mL and5×107CFU/mL. In the experiment, we used Shewanella spp.(KCCM41821),Edwardsiella tarda(ATCC15947), Aeromonas salmonicida(MT004), Fugustreptococcus(KCTC3657), Vibrio harveyi(ATCC14126), Vibrio splendidus(ATCC33125),Vibrio damsela(ATCC33539), Vibrio parahaemolyticus(KCTC2471), Vibrioalginolyticus(KCCM40513), Vibrio vulnificus(ATCC2126) and Vibrioanguillarum(KCTC2711). The strip had no cross-reaction with all the reference strains. Thestrip can be saved for6months at4℃or37℃. The test result are stable.The test strip developed in this study has high specificity, sensitivity and stability.Shewanella smarisflavi can be detected without professional instrument. The test strip issuitable for grassroots which can monitor Apostichopus japonicus disease caused byShewanella smarisflavi.