罗非鱼无乳链球菌巢式 PCR 检测方法的建立

摘要

Based on the cfb gene sequence of Streptococcus agalactiae, two pairs of primers were designed and synthesized and the amplification conditions were optimized to establish the nested PCR method which could identify S.agalactiae with high sensitively.The results showed that S.agalactiae could be directly detected from blood sample at a minimum concentra -tion as 8.7 ×10 4 CFU/ml by this method.Nineteen clinical S.agalactiae -infected tilapia samples collected from Guangxi province were applied to verify this method.The results showed that 260 bp fragment could be amplified in 18 samples and the sequencing analysis revealed that all the amplified fragment were cfb gene of S.agalactiae.The detection accuracy of this method was 94.7%.%根据无乳链球菌（Streptococcus agalactiae）cfb 基因序列，设计、合成2对引物，优化扩增条件，建立了快速高灵敏度鉴别无乳链球菌的巢式 PCR 方法。结果显示：使用该方法对罗非鱼（Oreochromis mossambicus）血液样品进行检测，可检测到活菌浓度为8.7×104 CFU／mL 的无乳链球菌。对采自广西地区受无乳链球菌感染的19份罗非鱼样品进行检测，18份可获得目的片段，扩增到的序列均为无乳链球菌 cfb 基因序列，检测准确度达到94.7％。