快速检测致病性副溶血弧菌的Dot-ELISA方法的建立

DOT-ELISA METHOD FOR RAPID DETECTION OF PATHOGENIC VIBRIO PARAHEMOLYTICUS

本研究选用硝酸纤维素膜作固相载体,以出现明显清晰斑点者判为阳性结果,成功建立了检测致病性副溶血弧菌耐热直接溶血素(direct heat hemolysin,TDH)的斑点酶联免疫吸附试验(Dot-ELISA)方法。根据棋盘试验,确定免疫血清最佳工作浓度为1:100,酶标二抗最佳工作浓度为1:1000。特异性检测结果表明,TDH阳性的副溶血弧菌可以呈现明显清晰斑点,而不能产生TDH的副溶血弧菌及其他对照菌(如嗜水气单胞菌、大肠杆菌、沙门氏菌和金黄色葡萄球菌)均无斑点显示。该方法与PCR方法的符合率为82.5%。本研究所建立的Dot-ELISA检测方法简便、快速,特异性和敏感性较高,适合基层和现场的初步筛选。

A dot-enzyme linked immunosorbent assay(Dot-ELISA)using nitrocellulose membrane as solid carrier was developed for detection of pathogen Vibrio parahaemolyticus.The samples with clear and visible dots were judged as positive reaction.The working concentrations determined by chessboard assay were 1:100 dilution for serum samples and 1:1000 dilution for HRP-conjugated antibodies.Vibrio parahaemolyticus strains with positive TDH were also positive in Dot-ELISA.No specific spots were visible in TDH negative bacteria,such as Vibrio fluvialis,Escherichia coli,Salmonella and Staphylococcus aureus.In addition,the Dot-ELISA method reached 82.5%agreement with PCR assay.Therefore,Dot-ELISA was suitable for primary screening of Vibrio parahaemolyticu as it was simple,rapid,sensitive and specific.