翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

MOLECULAR CHARACTERIZATION AND ESTABLISHMENT OF LAMP DETECTION METHOD OF PATHOGENIC AEROMONAS HYDROPHILA ISOLATED FROM SINIPERCA CHUATSI

  • 摘要: 嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。

     

    Abstract: Aeromonas hydrophila exists in the water body, which can cause many kinds of diseases and huge loss of aquaculture animals to the aquaculture production. The pathogenicity and virulence characteristics of the isolated strain which caused mass mortalities of reared Mandarin fish were studied. The rapid detection methods of A. hydrophila were established by the loop-mediated isothermal amplification (LAMP) using aerA gene as molecular marker. The results showed that the LD50 of A. hydrophila was 1.6×106 CFU/mL, and 14 virulence genes were detected from the isolated strain, which can be biology markers in detecting of pathogenic A. hydrophila. The assay of LAMP for detecting A. hydrophila was established using aerA gene as molecular marker. Positive reactions were detected by the stair-step amplified bands, and the green amplified products were observed directly by naked-eye in the reaction tube by addition of SYBR Green I. No any amplified bands and orange color were detected in control group. The results showed that LAMP detection method of A. hydrophila had good specificity. The results of sensitivity of LAMP showed that the primers could detect A. hydrophila at the level of 4.6×101 CFU/mL. The artificially infected 10 species of freshwater fishes and shrimps revealed positive amplified results using LAMP detection method, and no-infected 10 samples revealed negative amplified results. The results showed that the LAMP detection method has preferable application, and could be used in rapid diagnose of aquatic animal diseases caused by A. hydrophila.

     

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