超分支滚环扩增法检测美洲鳗鲡嗜水气单胞菌

Hyper-Branched Rolling Circle Amplification Assay for Detecting Aeromonas hydrophila in Anguilla rostrata

建立了基于锁式探针的超分支滚环扩增检测嗜水气单胞菌的新方法。根据嗜水气单胞菌ARE基因上的一段高保守基因序列,设计锁式探针和对应的扩增引物,并优化了反应条件。探针在T4 DNA连接酶作用下,16℃连接1 h、63℃扩增1 h,其扩增产物电泳后得到明显条带。在8种水产养殖病原菌中,只有嗜水气单胞菌可以特异性检出,并且特异性良好,灵敏度(1.0×10^(3) cfu/mL)高于免疫法以及常规的PCR法。对嗜水气单胞菌人工感染的美洲鳗鲡肌肉样品进行检测,发现条带清晰可见。该方法的反应过程是在恒温条件下进行,不需要PCR仪,此外,样品处理过程简单,应用水煮法即可进行。

A novel detection for Aeromonas hydrophila with hyper-branched rolling circle amplification(HRCA)based on padlock probe was established.A conservative sequence of the unique gene of ARE in A.hydrophila was used to designed the padlock probe and universal primer pair.The detection conditions were optimized.The result showed that padlock probes were linked with the target sequence of ARE at 16℃for 1 h by the T4 DNA ligase,then amplified at 63℃for 1 h.Finally,the strips of amplified products were effectively detected in electrophoresis.The strain of A.hydrophila was positive in HRCA,while 7 other contrast strains were negative in the assay,indicating that the assay had high specificity.The limit of detection was 1.0×10^(3) cfu/mL,and the sensitivity of this method was higher than that of immunoassay and conventional PCR.The HRCA was used to detect the muscle tissues from Anguilla rostrata infected artificial with A.hydrophila.The result indicated the strips were obvious.In the detection,the expensive PCR instrument was not needed because HRCA could be carried under a practically isothermal condition.Moreover,boiling-water-extraction of DNA in real samples simplified the steps,and the method had great potentials for the detection of bacteria in the fields of aquaculture.