摘要
为了建立一套用于实验室及室外现场检测迟钝爱德华菌(Edwardsiella tarda,Et)的环介导等温扩增方法(loop-mediated isothermal amplification,LAMP),以迟钝爱德华菌的毒力基因fimA为靶基因设计特异性引物,以基因组DNA为模板,进行环介导恒温扩增,并对其特异性、灵敏性和临床检测进行了试验。结果显示,迟钝爱德华菌阳性样本反应呈现为荧光绿色,阴性样本不变色。该LAMP方法的最适反应温度为63℃;特异性试验表明仅迟钝爱德华菌样本发生反应,而杀鲑气单胞菌、嗜水气单胞菌、温气单胞菌和豚鼠气单胞菌均不发生反应;敏感性试验表明,该LAMP方法可检出浓度为2.16×10^(-5)mg/L的迟钝爱德华菌的核酸。
In order to establish a loop-mediated isothermal amplification(LAMP)method for detection of Edwardsiella tarda in laboratory and field,a set of specific primers were designed based on the fimA gene sequence.Edwardsiella tarda DNA was used as a template for LAMP.The specificity,sensitivity and clinical detection were tested.The results showed that positive samples exhibited a fluorescent green color whereas negative samples exhibited a reddish brown color.The optimum temperature for the reaction was 63℃.Specific tests shown that the methed was capable to detect the Edwardsiella tarda with no cross action with Aeromonas salmon,Aeromonas hydrophila,Aeromonas Sobria and Aeromonas caviae.Sensitivity test showed that the methed could detect 2.16×10^(-5) mg/L Edwardsiella tarda DNA.
作者
何亚鹏
李小龙
刘鑫
张树林
杜迎春
李博
魏凯
邹强军
HE Yapeng;LI Xiaolong;LIU Xin;ZHANG Shulin;DU Yingchun;LI Bo;WEI Kai;ZOU Qiangjun(Beijing Aquatic wildlife rescue and conservation center,Beijing 102100,China)
出处
《水产养殖》
CAS
2023年第1期24-27,36,共5页
Journal of Aquaculture
基金
北京市鲟鱼、鲑鳟鱼创新团队项目(BAIC08-2021)。
