养殖大黄鱼病原弧菌多重PCR检测技术的建立和应用

Development and application of multiplex PCR of vibrios pathogenic to cultured large yellow croaker,Pseudnosciaena crocea (Richardson)

溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(Vibrio parahaemolyticus)和哈维氏弧菌(Vibrio harveyi)是浙江省养殖大黄鱼(Pseudnosciaena crocea)弧菌病的主要致病菌。本研究选择针对溶藻弧菌和副溶血弧菌的胶原酶基因,哈维氏弧菌的部分ToxR基因的特异性,优化设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,建立了一种检测致病性弧菌的多重PCR检测方法。经过琼脂糖凝胶电泳后的条带分析判断,可以在一个PCR管中同时成功地检测这3种病原细菌,含溶藻弧菌、哈维氏弧菌和副溶血弧菌3种致病弧菌核酸的阳性对照样品分别扩增出大小为737bp、382bp和271bp的预期产物,其灵敏度是102～103CFU/mL。将该方法应用于检测人工感染后的养殖大黄鱼病鱼肝脏和肾脏,结果在6份组织样本中,5份检出原始感染菌株,与API20E鉴定结果相符;对弧菌病流行季节采集的未发病的16份养殖大黄鱼组织样本和16份水体样本进行抽检,结果在1份大黄鱼组织样本中检出哈维氏弧菌,7份水体样本中检出这3种弧菌中的1种或2种,鉴定结果与API20E鉴定结果符合率为93.75%。说明该方法不仅可以检测发病鱼,还可以检测无病症带菌大黄鱼以及带菌水样,且说明海洋水体中存在着大黄鱼弧菌病的致病菌。结果说明,多重PCR检测方法具有较高的敏感性与特异性,可以缩短检测时间,降低检测成本,该方法的建立对养殖大黄鱼弧菌病的快速诊断和分子流行病学的调查具有重要意义。

In recent years,Vibriosis was the most severe disease of cultured large yellow croaker（Pseudnosciaena crocea） in Zhejiang province,China,resulting in great economic losses. The clinical symptoms included anorexia,floating upward and swimming alone,pterygiophore hyperaemia,ulceration,ascites and lippitude. It was epidemic from June to October, with peak on July to August. The mortality rate varied from 30 % to 40 % ,even up to 80% sometimes. It costed only one week from some fish infected to almost all dead. Three bacterial pathogens accounted for the majority of large yellow croaker vibriosis in Zhejiang province,China. They were Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio harveyi. In this study,three special oligonucleotide primers for multiplex PCR amplification was designed,based on the diversity of the collagenase gene sequences between Vibrio alginolyticus and Vibrio parahaemolyticus,and the partial ToxR gene specific detection of Vibrio harveyi. The reaction conditions of the multiplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of multiplex PCR were studied. Finally,the multiplex PCR is well developed successfully allowing the detection of the three bacterial pathogens in one PCR tube with relatively equal intensities DNA bands when analyzed in an agarose gel. Positive control samples which contained DNA of Vibrio alginolyticus,Vibrio harveyi and Vibrio parahaemolyticus yielded evident amplification products showing the expected DNA fragments of 737 bp,382 bp and 271 bp respectively. The sensitivity of multiplex PCR was 10^2-10^3 CFU/mL. When it was applied to detect the vibrios in livers and kidneys of fish which were artificially infected,the vibrio was detected in five of six samples,which was as same as the vibrio formerly injected. The result was coherence with that of API 20E method;when it was applied to detect the vibrios in different tissue samples of large yellow croaker and water samples collected from fishery in epidemic season,one of 16 tissue samples produced positive bands of Vibrio harveyi,and seven of 16 water samples produced positive bands of one or two vibrio species. The coherence tested by multiplex PCR and API 20E methods was 93.75%. The results showed that the multiplex PCR could be used not only to diagnose the clinically diseased large yellow croaker, but also to recognize the carrier. What is more,there are vibrios pathogenic to cultured large yellow croaker in the aquatic environment. It can be concluded that the multiplex PCR is specific and sensitive. It is a reliable tool for identification of the major Vibrios pathogenic with less time and cost,and it can be used in quick diagnose and epidemiology investgation of vibriosis of cultured large yellow croaker.