Detection of Nosema bombycis by FTA Cards and Loop-Mediated Isothermal Amplification (LAMP)

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• 作者：Wei Yan ; Zhongyuan Shen ; Xudong Tang ; Li Xu ; Qianlong Li ; Yajie Yue…
• 刊名：Current Microbiology
• 出版年：2014
• 出版时间：October 2014
• 年：2014
• 卷：69
• 期：4
• 页码：532-540
• 全文大小：706 KB
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• 作者单位：Wei Yan (1)
  Zhongyuan Shen (1) (2) (3)
  Xudong Tang (1) (2)
  Li Xu (1) (2)
  Qianlong Li (1) (2)
  Yajie Yue (1) (2)
  Shengyan Xiao (1) (2)
  Xuliang Fu (1)
  
  1. Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
  2. Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, 212018, Jiangsu, China
  3. Key Laboratory of Genetic Improvement of Silkworm and Mulberry of Agricultural Ministry, Zhenjiang, 212018, Jiangsu, China
  
• ISSN：1432-0991

文摘

We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10?spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100?% under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1?mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100?%. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24?h earlier than microscopy.