摘要:
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。
Abstract:
Two pairs of specific primers were designed and synthesized according to the 16SrRNA 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 305bp and 121bp were producted. Specificity test showed that two specific bands,16SrRNA 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 1.05×102 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.agalactiaein tilapia.
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