基于EB1基因的环介导等温扩增（LAMP）法检测感染家蚕微粒子病的蚕卵

【目的】家蚕 Bombyx mori 微粒子病一直影响着蚕种业的健康发展，快速而准确地检测出寄生于蚕卵中的家蚕微孢子虫 Nosema bombycis 对于有效控制家蚕微粒子病危害意义重大。【方法】环介导等温扩增（loop-mediated isothermal amplification, LAMP）法是一种快速、灵敏、特异的DNA体外恒温扩增方法，本文基于LAMP检测法的原理，依据家蚕微孢子虫孢子繁殖复制相关的 EB1 基因（GenBank登录号：KF421134.1）设计LAMP引物，对LAMP反应体系的最佳反应温度、内外引物浓度比、检测反应的特异性、灵敏度等进行研究，建立了一种检测蚕卵感染家蚕微粒子病的LAMP检测方法。【结果】结果表明，基于EB1 基因建立的LAMP检测方法在63℃恒温下在1.5 h内就可完成对样品的有效检测，本LAMP法对家蚕微孢子虫孢子DNA的检测灵敏度为5.0×10^-3 ng/μL，对EB1-pMD^TM19-T质粒标准品的检测灵敏度为1.0×10² copies/μL，同时对人工感染家蚕微粒子病单个母蛾产下蚕卵的1/8卵圈量或1粒蚕卵均能检出阳性结果。上述结果都分别应用凝胶电泳法、恒温荧光检测仪以及SYBR GreenⅠ显色肉眼观察法得到同步判定。【结论】本研究建立的基于EB1基因LAMP法可用于蚕卵微粒子病的检测，LAMP法为蚕种质量检验及成品卵微粒子病现场检疫提供了新技术。

Detection of pebrine disease in Bombyx mori eggs with the loop-mediated isothermal amplification (LAMP) method based on EB1 gene

【Aim】 Silkworm pebrine disease has been affecting the healthy development of the silkworm eggs’ industry. It is of great significance to effectively control the harm of pebrine disease via rapidly and accurately detecting the silkworm eggs parasitized by pebrine. 【Methods】 Loop-mediated isothermal amplification (LAMP) is a method that amplifies the template DNA with rapidity, sensitivity and high specificity under constant temperature in vitro. Based on the LAMP protocols, we selected Nosema bombycis EB1 gene (GenBank accession no. KF421134.1), a gene which is related to the replication of spores, to design the sets of LAMP primers. We investigated the optimal reaction conditions of the LAMP system, including the reaction temperature and the concentration ratio of the internal primer to outer primer, and detected the specificity and sensitivity of the LAMP reaction system. We also set up the LAMP assay for detecting the pebrine disease in silkworm eggs. 【Results】 LAMP assay based on EB1 could effectively detect the silkworm eggs infected with N. bombycis.The detection could be completed within 1.5 h at the constant temperature of 63℃. It was able to detect as low as 5.0×10^-3 ng/μL of the template DNA extracted from N. bombycis spores, and 100 copies of the constructed standard plasmid EB1-pMD^TM 19-T. Furthermore, it could also effectively detect 1/8 of the eggs batch from one infected silkworm moth by pebrine or one infected silkworm egg. The results were simultaneously demonstrated by running the gel electrophoresis, and by observing the fluorescence indicator, and also by visual observation with SYBR Green I, respectively. 【Conclusion】 The LAMP assay based on EB1 gene of N. bombycis for detecting the silkworm eggs infected by pebrine has been successfully established. It is further proved that the LAMP method could be used for the quality inspection in production of silkworm eggs or for quarantine inspection of the commercial eggs on sites.