日本对虾(Penaeus japonicus)病原需钠弧菌(Vibrio natriegens)表型与分子特征及LAMP检测方法的建立

采用常规表观生物学特性及16S rRNA、gyrB及rpoA基因同源性检索与系统发育学分析等方法,对分离自江苏连云港某育苗场的大批死亡日本对虾蚤状幼体的优势生长菌进行了综合鉴定。结果表明,其形态和生理生化特征与弧菌属的需钠弧菌相近;16S rRNA、gyrB及rpoA基因同源性检索也均与需钠弧菌相似性最高,分别为98%、89%和95%,且三种基因的NJ系统发育树也均与需钠弧菌聚为一个分支;分离菌的致病性试验表明其半数致死量LD50为1.8×106CFU/ml。综合分离菌的致病性、形态与生理生化特征及基因同源性与系统发育分析结果,认为引起日本对虾蚤状幼体大批死亡的病原为需钠弧菌。基于gyrB基因序列设计1套LAMP特异性引物,建立了需钠弧菌的快速特异性检测方法,可用于由需钠弧菌引起的水产动物疾病的诊断及分子流行病学的调查研究。

PHENOTYPIC AND MOLECULAR CHARACTERIZATION AND THE LAMP DETECTION OF PATHOGENIC VIBRIO NATRIEGENS ISOLATED FROM DISEASED PENAEUS JAPONICUS

A mass mortality of zoea of Penaeus japonicus broke out in a hatchery in Lianyungang, Jiangsu Province. The dominant bacterial colonies were isolated and confirmed the pathogenicity by challenge experiments, the LD₅₀ of the isolates to P. japonicus was 1.8×10⁶ CFU/ml. The morphological, physiological, biochemical characteristics were examined, and the 16S rRNA gene, gyrB gene and rpoA gene sequences were amplified and compared with sequences stored in the databases, from which molecular phylogenetic trees were constructed. The isolates showed that properties and biochemical characteristics similar to Vibrio natriegens. The sequenced 16S rRNA gene of strain JL120508 (GenBank accession No.JX993271) was 1461bp, the gyrB gene of strain JL120508 (GenBank accession No.JX993272) was 1179bp, and the rpoA gene of strain JL120508 (GenBank accession No.JX993273) was 1038bp in length. The 16S rRNA, gyrB gene and rpoA gene sequences displayed high similarity to the genes of V. natriegens from GenBank database. The phylogenetic trees based on 16S rRNA, gyrB and rpoA sequences, JL120508 clustered with the V. natriegens strains, and was supported by high bootstrap value, strongly supporting the assignment of isolate JL120508 to V. natriegens. In addition, specific primers were designed based on the gyrB gene sequences, and the loop-mediated isothermal amplification (LAMP) detecting V. natriegens was established. The LAMP is suggested to be useful in the specific and rapid diagnose and epidemiological investigation of the disease caused by V. natriegens.