Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs
Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs
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开发用于快速、视觉检测蚕卵中家蚕微孢子虫的核酸侧流试纸条
DOI：
10.1016/j.jip.2019.04.004
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发表时间：
2019-06-01
影响因子：
3.4
通讯作者：
Pan, Guoqing
中科院分区：
文献类型：
--
作者：
He, Zhangshuai;Ni, Qi;Pan, Guoqing
家蚕微粒子病的病原家蚕微孢子虫给蚕业造成了巨大的经济损失。因此，商业蚕种的检疫是对桑蚕业的重要保障。在这里，我们建立了一个基于聚合酶链式反应(PCR)和胶体金试纸条相结合的核酸横向流动条(NAFLS)的用户友好的检测系统。根据家蚕微孢子虫LSU rDNA序列设计了一对对家蚕常见微孢子虫分离株具有良好特异性的聚合酶链式反应引物。正向和反向引物在5‘端分别用生物素和羧基荧光素(FAM)标记。从鸡蛋样品中提取基因组DNA作为模板进行聚合酶链式反应，然后用NALFS进行检测。纯化的家蚕基因组DNA的检测下限为1pg，比琼脂糖凝胶电泳法(AGE)高100倍。此外，对模拟感染的蚕卵的检测灵敏度比年龄高10-100倍。在29份实验室感染雌蛾产卵的蚕卵样品中，NALFS检出感染的有27份；每批感染卵检出阳性率为2%，每批感染卵检出40%的虫卵才能进行年龄检测。综上所述，NALFS方法简单易行，在蚕种生产中检测家蚕微孢子虫具有广阔的应用前景。
Nosema bombycis, the pathogen of silkworm pebrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5' end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100 x more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10-100 x higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; >= 2% infected eggs per batch are detected as positive, while >= 40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production.