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Rapid detection of two pathogens of skin ulcer syndrome in cultured Apostichopus japonicus by Dot-ELISA

XIE Jian-jun1,2,WANG Yin-geng1,ZHANG Zheng1,RONG Xiao-jun1(1.Key Laboratory for Sustainable Utilization of Marine Fisheries Resources,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;2.Biology Science&Technology College,Shanghai Fisheries University,Shanghai 200090,China)  
A Dot-ELISA test has been developed for the rapid detection of Vibrio splendidus and Pseudoalteromonas nigrifaciens that were the two pathogens of the skin ulcer syndrome in sea cucumber Apostichopus japonicus.The antisera of V.splendidus and P.nigrifaciens were prepared with rabbits.The best working concentrations of two kinds of antisera and HRP-goat-anti-rabbit IgG(HRP-IgG) were assayed by chessboard test.As V.splendidus was detected,the dilution ratios of antiserum and HRP-IgG were 1∶320 and 1∶3 000.As to P.nigrifaciens,the dilution ratios were 1∶160 and 1∶2 000.Cross-reactions of antiserum with other main pathogens were examined,and the results showed that low cross reaction could be mostly eliminated by heightening the dilution of antiserum.The cross-reaction assay and absorb assay indicated the method was successful in distinguishing those pathogens.The established method was able to detect the pathogens from diseased sea cucumber,water and even normal sea cucumber in infection test.The sensitivity of the method was 9.4×103 cells/mL.The same results were also verified by the direct agglutination test.This study showed that Dot-ELISA was a rapid detection method,and because of its ease of performance and high specificity and no instrumentation required,it could be applied under field conditions where sophisticated facilities are lacking.
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