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为建立准确快速的海豚链球菌鉴定方法,设计合成了海豚链球菌种特异性引物CM1/CM2,进行了其特异基因片段的PCR扩增、反应条件的优化及方法的特异性和敏感性试验;同时还进行了不同检测材料的比较及9份临床样品检测。结果表明,引物CM1/CM2只能从海豚链球菌中扩增到特异性基因片段,供试的其它9种水产常见病原菌PCR扩增均呈阴性;能够检测的最低细菌数在20~30个细菌;方法可直接从病鱼的脑、肝脏、肾脏及脾脏组织检测到该菌;另外,临床菌株检测结果与基于菌株16S rRNA基因序列系统进化分析结果一致。该方法弥补了传统细菌鉴定很难将该菌鉴定到种的缺点,并显著缩短了检测时间及降低了检测成本,具有较好的应用前景。
In order to establish an accurate and rapid method for the identification of Streptococcus suis, we designed and synthesized the species-specific primers of Streptococcus viridans CM1 / CM2, carried out PCR amplification of its specific gene fragments, optimization of reaction conditions and specificity and sensitivity tests; At the same time also conducted a comparison of different test materials and 9 clinical samples. The results showed that the primer CM1 / CM2 could only amplify the specific gene fragment from Streptococcus pulex puffer, and the other 9 common pathogenic bacteria of aquatic organisms tested were negative for PCR amplification. The lowest number of bacteria that could be detected was between 20 and 30 bacteria . The method could detect the bacteria directly from diseased fish brain, liver, kidney and spleen. In addition, the results of clinical strains were consistent with the phylogenetic analysis based on the 16S rRNA gene sequences of strains. The method can make up for the shortcomings that the traditional bacteria identification is hard to identify the bacterium to the species, and the detection time is shortened and the detection cost is reduced, and the method has good application prospect.