牡蛎单孢子虫环介导等温扩增可视化检测方法的建立

目的:建立基于环介导等温扩增(LAMP)技术的单孢子虫可视化检测方法。方法:根据尼氏单孢子虫的小亚基核糖体RNA保守序列,设计一套特异性LAMP引物,对反应条件如温度和试剂浓度进行优化,建立检测牡蛎单孢子虫的LAMP方法。结果:所建立的方法的敏感性可达1 fg,是常规PCR方法的100倍;全部反应可在1 h内完成;可通过肉眼观察颜色,直接判定结果;对其他牡蛎常见病原体的检测结果均为阴性。结论:建立的LAMP方法简便、快速、灵敏、特异,可用于牡蛎单孢子虫感染的快速检测。

Development of a Loop-Mediated Isothermal Amplification Assay for Visual Detection of Haplosporidium in Oyster

Objective： To develop a loop-mediated isothermal amplification（LAMP） assay for detection of Haplosporidium sp.Methods： According to the small subunit ribosomal RNA sequence of H.nelsoni in GenBank,six primers were designed to be used in LAMP assay.The reaction parameters such as the concentration of the buffer and the reaction temperature were optimized to develop the LAMP assay for detection of Haplosporidium sp.in oyster Results： The detection limit of this LAMP method was 1 fg,and it was much more sensitive than routine PCR assay.The amplification could be finished within 1 h,and the presence of Haplosporidium sp.could be detected by naked eyes.There was no cross-reactivity with the other pathogen of oyster.Conclusion： This LAMP assay is a simple and specific method for rapid detection of Haplosporidium sp.in clinical samples.