红尾皇冠鱼无乳链球菌病LAMP检测方法的建立与应用

Development and application of loop-mediated isothermal amplification for detection of Streptococcus agalactiae in green terror Aequidens rivulatus

以cfb基因保守序列的6个区域设计4条特异性引物,利用链置换DNA聚合酶（Bst DNA polymerase）在恒温（65℃）下保温40 min,建立了无乳链球菌Streptococcus agalactiae的LAMP技术。建立的LAMP方法能够特异性地检测无乳链球菌,检测灵敏度为3.7×10^1～3.7×10^2cfu/mL,反应前加入显色剂钙黄绿素避免二次污染。现场应用中采用FTA卡采集红尾皇冠鱼Aequidens rivulatus病鱼肝脏组织病原菌,操作简便、快捷。研究表明,针对无乳链球菌cfb基因建立的LAMP检测方法具有较高的特异性及稳定性,尤其是具有快速、简便、成本低等优点,可用于无乳链球菌的野外现场快速检测。

The cfb gene of Streptococcus agalactiae was amplified by a set of four specific primers that recognize six distinct sequences of the target in 40 min by incubating all of the reagents in a single tube with Bst DNA polymerase at a constant 65 ℃. A loop-mediated isothermal amplification（ LAMP） assay was developed and validated for the specific detection of Streptococcus agalactiae,with detection limits of 3. 7 × 10^1-3. 7 × 10^2 cfu / mL. The resulting amplicons were visualized by adding calcein to the reaction tube before the reaction. FTA cards was used for collection and purification of nucleic acids from a liver of green terror Aequidens rivulatus with pathogenic bacteria in field applications. The findings demonstrate that the LAMP-based assay is a sensitive and reliable means for the detection of Streptococcus agalactiae with rapidity,simplicity and low cost,especially suitable for on-site rapid diagnosis.