Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia
Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia
复制标题
DOI：
10.1111/j.1365-2109.2009.02214.x
复制
发表时间：
2009-07-07
影响因子：
2
通讯作者：
Lin, King-Jung
中科院分区：
文献类型：
--
作者：
Chang, Chin-I;Wu, Chia-Che;Lin, King-Jung
建立了一种基于多重套式聚合酶链式反应(m-套式聚合酶链式反应)的方法，用于同时检测亚热带亚洲淡水/海水鱼类的4种重要病原菌，包括嗜水气单胞菌、迟缓爱德华氏菌、淡色光杆菌和新链球菌。证实了用于检测的寡核苷酸引物的特异性，可以对相应的病原体产生特异的扩增产物。此外，从23种罗非鱼或罗非鱼组织匀浆的31个相关菌株中提取的纯DNA进行检测时，还观察到了非特异性扩增。M-套式聚合酶链式反应可检测到嗜水气单胞菌19个菌落形成单位(CFU)、迟缓艾美耳球虫62个菌落形成单位(CFU)、粉纹夜蛾亚种280个菌落单位(CFU)。在感染罗非鱼肾组织匀浆中检出鱼纲和179 CFU，与细菌学方法检测结果一致。该方法是一种灵敏、有效的同时检测多种鱼类病原体的方法。
A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.