利用LAMP技术对蝌蚪养殖池水霉菌的检测

Rapid Detection of Saprolegnia in Water of Tadpole Feeding-Pound by Loop-Mediated Isothermal Amplification

水霉病由水霉菌引起,能感染包括鱼类、两栖类在内的多种水生动物,严重损害水产养殖行业的经济利益。由于水霉菌具有多宿主、生命力顽强的特点,及时监测水体中水霉菌及其孢子的浓度成为预防水霉病的重要方面。本研究利用环介导等温扩增技术(LAMP)建立了水霉菌LAMP检测方法,并在实际生产中进行了临床应用。结果表明:在链置换聚合酶(Bst酶)的作用下,62℃扩增60 min,扩增结果可用琼脂糖凝胶电泳或荧光染料两种方式观察得到,最低可检出102个拷贝,具有较高的特异性。利用该方法对某林蛙养殖场的蝌蚪池水霉菌进行检测,结果为阳性,证明该养殖场的蝌蚪池中存在水霉病暴发和传播的风险。

Saprolegniasis caused by Saprolegnia can infect a variety of aquatic animals including fish,amphibians,and causes serious damage to the economic interests of the aquaculture industry.Because of the Saprolegnia characteristics,such as multi-host and good vitality,the important aspect of preventing Saprolegniasis is to monitor the concentration of Saprolegnia and its spore without delay. In this study,the Saprolegnia LAMP detection method using a loop mediated isothermal amplification（ LAMP） was established and conducted the clinical application in the actual production. The experimental results showed that the reaction of LAMP was completed within 60 min by Bst polymerase catalysis under isothermal condition of 62℃. Through the optimization of the experimental parameters,the number of the minimum detectable amplification could be up to 10^2 copies of Saprolegnia,which had a high specificity. Using the method to detect the Saprolegnia in a tadpole feeding-pound of Rana dybowskii farms,the result was positive,which meaned this farm existed the risk of Saprolegniasis outbreaking and spreading.