养殖刺参腐皮综合征两种致病菌Dot-ELISA快速检测

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

以养殖刺参(Apostichopus japonicus)腐皮综合征两种主要致病原灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)为抗原,分别制备兔抗血清,建立了两种病原菌的点酶ELISA(Dot-ELISA)检测方法。根据方正试验,确定检测用一抗、二抗最佳工作稀释度:灿烂弧菌抗血清稀释度320,酶标二抗稀释度3 000;假交替单胞菌抗血清稀释度160,酶标二抗稀释度2 000。阻断试验结果表明两种抗血清特异性均较高。灿烂弧菌抗血清在稀释度为40时与溶藻胶弧菌、河流弧菌、创伤弧菌等弧菌发生微弱交叉反应,提高稀释度到160时,不发生交叉反应。假交替单胞菌抗血清基本不与常见海水致病弧菌发生交叉反应。人工感染试验检测结果表明该方法能从患病海参溃烂组织、未发病海参组织和感染水体检测到相应致病菌,检测灵敏度为9.4×103个/mL。人工感染试验检测结果经直接凝集反应验证。该方法操作简便、灵敏度高,不需复杂仪器设备,适合在基层推广。

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 antise rum 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 × 10^3 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.