Species-specific polymerase chain reaction primers for Lactococcus garvieae
Species-specific polymerase chain reaction primers for Lactococcus garvieae
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DOI：
10.1046/j.1365-2761.2000.00207.x
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发表时间：
2000-01-01
影响因子：
2.5
通讯作者：
Hirono, I
中科院分区：
文献类型：
--
作者：
Aoki, T;Park, CI;Hirono, I
从鱼类致病菌Lactococcus garvieae（以前称为Enterococcus seriolicida）的染色体DNA中克隆了一个二氢蝶酸合酶基因。然后选择该基因作为聚合酶链反应（PCR）的靶基因。所设计的PCR引物仅扩增出一个709 bp的片段。garvieae菌株中扩增出相同大小的片段，而L.乳酸肠球菌、粪肠球菌、E.粪链球菌或β-溶血性链球菌。在TNES-Urea中储存了Garvieae和健康黄尾鱼的肾组织样品。通过标准方法的修改和煮沸提取方法从组织样品中提取DNA。特别是，在通过煮沸提取法提取和纯化后30分钟内利用模板DNA。这些种特异性PCR引物能扩增出一株L.从自然感染L. Garvieae。本文介绍了L.从DNA提取到电泳后在琼脂糖凝胶中观察，鱼中的Garvieae感染可以在不到4小时内进行。
A dihydropteroate synthase gene from the chromosomal DNA of the fish-pathogenic bacteria Lactococcus garvieae (formerly Enterococcus seriolicida) was cloned. This gene was then chosen as the target for polymerase chain reaction (PCR). The designated PCR primer set only amplified a 709-bp DNA fragment from L. garvieae strains, and did not amplify the same molecular size fragment from related species of L. lactis, Enterococcus faecalis, E. faecium or beta-haemolytic Streptococcus sp. The kidney tissue of yellowtail, Seriola quinqueradiata (Temminck and Schlegel), a species which is naturally infected with L. garvieae, and also kidney tissue samples of healthy yellowtail were stored in TNES-Urea. The DNA was extracted from tissue samples by a modification of the standard method and by a boiled-extraction method. In particular, template DNA was utilized within 30 min following extraction and purification by the boiled-extraction method. These species-specific PCR primers could amplify a L. garvieae target sequence from yellowtail which were naturally infected with L. garvieae. The total procedure for the diagnosis of L. garvieae infections in fish, from the point of DNA extraction to observation in an agarose gel following electrophoresis, can be performed in less than 4 h.