Results of approbation of three pairs of primers for identification of the causative agent of salmon furunculosis Aeromonas salmonicida by PCR

A. V. Polteva, E. V. Galanina, D. A. Viktorov, A. A. Lomakin
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Abstract

The aim of the work was to select and test several pairs of primers to identify the causative agent of A. salmonicida salmon furunculosis by PCR.The methods: six bacterial cultures isolated from pre-spawning chum salmon with and without external signs of furunculosis, caught in rivers in the south of Sakhalin, were used as test cultures during the testing of primers. Ready-made kits D-Cells‑250 and Intifica TaqM master mix were used to isolate bacterial DNA and prepare PCR mixtures. Amplification was carried out in a thermocycler T‑100 ThermoCycler (Bio-Rad). Detection of PCR products was carried out by electrophoresis in 1.5% agarose gel in triacetate buffer (TAE). To view the results and document them, the Bio-Rad Gel DOC XR+system was used.The results: the species specificity of three pairs of primers selected for the identification of the causative agent of furunculosis by PCR was experimentally confirmed. Testing of the selected primers was carried out on bacterial cultures isolated from pre-spawning chum salmon individuals with and without manifestations of furunculosis. According to the results of testing, all bacterial isolates were assigned to the species A. salmonicida.Novelty of the work: for the first time, a comparison of primers proposed by several authors for the identification of the causative agent of furunculosis was performed on bacterial cultures isolated from salmon of the Far Eastern region.Practical significance: the obtained results were used to prepare methodological guidelines for the identification of the causative agent of salmon furunculosis A. salmonicida by PCR, which reduces the time of diagnosis of the disease.
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通过 PCR 鉴定鲑鱼毛滴虫病病原体的三对引物的批准结果
方法:在测试引物时,使用了从萨哈林岛南部河流中捕获的产卵前大马哈鱼身上分离出的六种细菌培养物作为测试培养物,这些培养物有的患有大马哈鱼疖病,有的没有疖病的外部症状。使用现成的 D-Cells-250 试剂盒和 Intifica TaqM 母液来分离细菌 DNA 和制备 PCR 混合物。扩增在热循环仪 T-100 ThermoCycler(Bio-Rad)中进行。PCR 产物在三醋酸缓冲液(TAE)中的 1.5%琼脂糖凝胶中电泳检测。使用 Bio-Rad Gel DOC XR+ 系统查看和记录结果。结果:实验证实了用于通过 PCR 鉴定疖病病原体的三对引物的物种特异性。对所选引物的测试是在从产卵前大马哈鱼个体中分离出的细菌培养物上进行的,这些个体有的患有毛囊虫病,有的则没有。工作的新颖性:首次在从远东地区鲑鱼分离的细菌培养物上对多位作者提出的用于鉴定鲑鱼疖病病原体的引物进行了比较。实用意义:利用所获得的结果制定了通过 PCR 鉴定鲑鱼疖病病原体 A. salmonicida 的方法指南,从而缩短了疾病的诊断时间。
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