Abstract: 【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.

Key words: Bombyx mori, pebrine disease; Nosema bombycis, loop-mediated isothermal amplification, fluorescence indicator, gel electrophoresis, PCR