【作者】 苏友禄； 李言伟； 李安兴；

【Author】 You-Lu Su;Yan-Wei Li;An-Xing Li;Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol,The School of Life Sciences,Sun Yat-sen University;

【机构】 中山大学生命科学学院水产品安全教育部重点实验室/生物防治国家重点实验室；

【摘要】 近年来,罗非鱼无乳链球菌病大范围爆发流行,对罗非鱼养殖造业成巨大损失。目前,虽然在无乳链球菌毒力基因、致病性和基因组学等方面取得一定的研究进展,但该病原菌在宿主体内的定植规律尚未明确。本实验根据无乳链球菌16S-23S r DNA间区序列设计特异性引物,通过反应条件优化、敏感性及特异性测试,成功构建该菌的荧光定量PCR检测方法,其检测限可达8.6个菌拷贝/20μL的反应体系。在人工感染条件下,检测到无乳链球菌在罗非鱼组织内的定植规律为:脑组织内菌载量最高;其次为眼、肠、脾、心和肾;肝、鳃和肌肉中最低。此外,用不同毒力的无乳链球菌感染罗非鱼,检测各菌株在感染恢复期罗非鱼脑组织内的定植情况,结果表明,其菌载量在罗非鱼脑组织内差异不显著。更多还原

【Abstract】 Streptococcus agalactiae has become one of the most important emerging pathogens in aquaculture industry, especially, causing large economic losses in tilapia farms of China. Although the pathogenicity and genome of S. agalactiae has been ascertained, bacterial load in infected tilapia remains to be investigated. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions(q PCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a targeting the16S-23 S r RNA intergenic spacer regions was finally chosen with a detection limit of 8.6 copies of S. agalactiae in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by q PCR from the tissues of Oreochromis niloticus at 5 days post injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, and with higher levels in kidney, heart, spleen, intestines and eye, while with lower levels in muscle, gill and liver. Additionally, q PCR results indicated that lower bacterial loads with no significant differences were observed in the brain of convalescent O. niloticus at 14 days post injection with different S. agalactiae strains. This approach provides a measurable tool for studying bacterial tissue tropism in infected fish, as well as, for monitoring bacterial colonization in the convalescent fish.更多还原