双重PCR检测罗非鱼源无乳链球菌方法的建立

Rapid Detection of Stretococcus agalactiae Isolated from Tilapia with Double PCR

根据罗非鱼源无乳链球菌16SrRNA基因和sip基因序列,设计、合成2对特异性引物,通过对反应体系和反应条件优化,建立快速检测无乳链球菌的双重PCR方法。该方法扩增无乳链球菌可获得1 305bp和121bp 2个特异性片段;对6株链球菌属的菌株进行特异性分析,仅无乳链球菌能扩增出16SrRNA基因和sip基因2条特异性条带,而海豚链球菌无条带检出;经灵敏度试验,可检测到无乳链球菌菌株070717LL的最小量为1.05×102 CFU;同时,双重PCR可以检测出无乳链球菌菌株070717LL人工感染的罗非鱼脾、脑、肾组织中细菌DNA,特别是脾脏和肾脏组织的样品检测效果好。结果证明所建立的方法具有快速、特异性强、灵敏度高等优点,适用于对罗非鱼源无乳链球菌病的流行病学调查。

Two pairs of specific primers were designed and synthesized according to the 16S rRNA and Surface immunogenic protein （sip） gene sequence of tilapia Stretococcus agalactiae ,and a double-PCR method for rapid detection of Streptococcus agalactiae isolated from tilapia was established through optimization of reaction system and reaction condition ,by which two specific fragments for S .agalactiae of 1 305 bp and 121 bp were producted . Specificity test showed that two specific bands ,16S rRNA and sip ,could only be detected in the S . agalactiae , and none was amplified from Streptococcus inia .The minimum detectable quantity of the S . agalactiae strains 070717LL was 1.05 ＆#215; 10^2 CFU . In the tissues from spleen , brain and kidney of tilapias affected by the S . agalactiae strain 070717LL the strains DNA were detected , especially in kidney and spleen tissues . In conclusion ,this double-PCR method was specific ,sensitive rapid ,and applicable for epidemiological investigation of S . agalactiae in tilapia .