Yan Wei, Shen Zhongyuan, Tang Xudong, Xu Li, Li Qianlong, Yue Yajie, Xiao Shengyan, Fu Xuliang
Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China,
Curr Microbiol. 2014 Oct;69(4):532-40. doi: 10.1007/s00284-014-0619-3. Epub 2014 Jun 4.
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.
我们通过结合玻璃珠、FTA卡和环介导等温扩增技术（LAMP）成功建立了一种检测方法，该方法对家蚕微孢子虫的检测灵敏度明显高于先前开发的检测方法。家蚕微孢子虫的孢子首先用酸洗玻璃珠破碎；随后用FTA卡提取并纯化DNA，并使用基于家蚕微孢子虫 LSU rRNA序列设计的引物（LSU296）进行LAMP反应。最低检测浓度为10个孢子/毫升。当使用该方法检测蚕卵中的微粒子病时，在我们优化的条件下，对于500粒蚕卵（其中只有一粒卵感染了家蚕微孢子虫）的检测率为100%。如果样品中的卵数增加到800或1000，将样品分成两等份，并在加入1毫升TE缓冲液后用玻璃珠捣碎蚕卵。随后将两管中的液体混合并应用于FTA卡，检测率为100%。此外，我们研究中建立的LAMP方法比显微镜检查能提前24小时检测到家蚕微孢子虫感染。
