环介导等温扩增法检测熊蜂中的微孢子虫

Detection of Microsporidia in Bumble Bees by Loop-mediated Isothermal Amplification

为快速检测进口熊蜂中的微孢子虫,建立了环介导等温扩增技术(Loop-mediated isothermal amplifi cation,LAMP)。针对熊蜂微孢子虫核糖体保守基因设计LAMP引物,以熊蜂微孢子虫DNA、家蚕微孢子虫DNA、蝗虫微孢子虫DNA、兔脑炎微孢子虫DNA作为LAMP反应的模板,分别采用浊度法和显色法验证LAMP引物的特异性;并将含微孢子虫基因的质粒模板10倍梯度稀释,考察方法的敏感性。结果表明,LAMP法能有效、特异地检测出熊蜂寄生微孢子虫DNA,灵敏度比PCR法高10倍。该方法简单、快速、敏感,可应用于熊蜂寄生微孢子虫的便捷检测中。

In order to rapidly detect Microsporidia in imported bumble bees, a method of loop-mediated isothermal amplification （LAMP） was developed. Firstly, the LAMP primers were designed according to the conservative gene of Microsporidia ribosome in bumble bees, then through different DNA templates including Nosema bombi, Nosema bombycis, Nosema Iocustae and Encephalitozoon cuniculithe, the specificity of this LAMP method was verified by both turbidimetric and color-developing method. Meanwhile, its sensibility was investigated by using plasmid templates ofNosema bombi gene of 10-fold serial dilution. It＇s showed that the developed LAMP method could detect Nosema bombi DNA effectively and specifically, and its sensitivity was ten times higher than that of PCR method. Due to the characteristics of simplicity, speediness and accuracy, this method could be applied to the detection of Microsporidia in bumble bees.