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首页> 外文期刊>Aquaculture >Use of random amplification to develop a PCR detection method for the causative agent of fish pasteurellosis, Photobacterium damselae subsp piscicida (Vibrionaceae)
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Use of random amplification to develop a PCR detection method for the causative agent of fish pasteurellosis, Photobacterium damselae subsp piscicida (Vibrionaceae)

机译：利用随机扩增开发一种PCR检测方法，用于检测鱼巴氏杆菌病（Pasper damselae subsp piscicida（Vibrionaceae））的病原体

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Random amplification of polymorphic DNA (RAPD) was used to generate species-specific markers, which were exploited in the construction of a fast and convenient PCR-based diagnostic assay for the causative agent of fish pasteurellosis or pseudotuberculosis, the bacterium Photobacterium damselae subsp. piscicida, formerly known as Pasteurella piscicida. In RAPD genomic fingerprints of this bacterium, two distinctive fragments were observed. These fragments were cloned, sequenced and found to bear no significant homology with known DNA sequences. Two sets of primers were then synthesized to amplify, by PCR, fragments within the sequences between the terminal RAPD primers of the fragments. Another primer pair was designed to perform nested PCR internally to one of the amplified fragments. The species specificity of the PCR products was analyzed, by dot blotting and Southern hybridization, with DNA from 13 other bacterial species. Cross-reactivity was obtained only with isolates of P. damselae subsp. damselae (formerly Vibrio damselae) a bacterial species with a high degree of overall genomic homology with P. d. piscicida. The detection limit of the assay was 20 fg of template DNA by PCR and 2 fg by nested PCR. The assay was effective in revealing P. d. piscicida when applied to an aliquot of DNA extracted from 50-mg samples of kidney, spleen and liver tissues from naturally infected fish. In conclusion, these results demonstrate the utility of the RAPD plus PCR approach in the development of genetic diagnostic assays for fish pathogens.
机译：多态性DNA（RAPD）的随机扩增用于产生物种特异性标记，这些标记被用于构建基于快速，便捷的基于PCR的鱼巴氏杆菌病或假结核病致病菌，即淡水梭菌亚种亚种的病原体诊断方法。 piscicida，原名巴斯德氏杆菌。在该细菌的RAPD基因组指纹图中，观察到两个独特的片段。这些片段被克隆，测序并发现与已知的DNA序列没有明显的同源性。然后合成两组引物，以通过PCR扩增片段的末端RAPD引物之间的序列内的片段。设计另一对引物以对一个扩增片段内部进行巢式PCR。通过斑点印迹和Southern杂交，用来自其他13个细菌物种的DNA分析了PCR产物的物种特异性。仅与damselae亚种的亚种分离得到交叉反应。 damselae（damselae）（原弧菌）。与P. d。高度基因组同源的细菌种。皮西奇达。该测定的检测限是通过PCR PCR模板为20 fg，通过嵌套PCR PCR模板为2 fg。该测定法有效地揭示了P.d。 piscicida应用于从50毫克天然感染鱼的肾脏，脾脏和肝脏组织样品中提取的DNA等分试样时。总之，这些结果证明了RAPD加PCR方法在开发鱼类病原体的基因诊断分析方法中的实用性。

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