鸡毒支原体强弱毒株LAMP可视化鉴别检测方法的建立

为建立一种能鉴别鸡毒支原体强弱毒株的快速检测方法,根据GenBank中鸡毒支原体S6株的16SrRNA基因序列和F弱毒疫苗株假定的α磷酸海藻糖酶基因序列,设计2套环介导等温扩增引物,建立了鸡毒支原体强弱毒株的环介导等温扩增可视化鉴别检测方法。该方法的敏感性可达10fg,是常规PCR方法的100倍,对其他鸡常见病原体的检测结果均为阴性。全部反应只需一个可控温的水浴锅在1h内即可完成,通过肉眼观察颜色直接判定结果。本研究建立的环介导等温扩增方法简便、快速、灵敏、特异,是一种适于基层工作站的鸡毒支原体强、弱毒株鉴别检测方法。

Development of a loop-mediated isothermal amplification assay for visual differentiation of Mycoplasma gallisepticum virulent strains from attenuated strains

The aim of the present study was to establish a rapid method for differentiation of the virulent strains and the attenuated strains of Mycoplasma gallisepticum(MG).Based on the conserved sequences of the 16 S rRNA gene of the virulent strain S6 and putative alpha-phosphotrehalase gene of the vaccine strain F of MG from GenBank,two sets of the loop-mediated isothermal amplification(LAMP) primers were designed to amplify the target genes.The LAMP assay was established to visually differentiate the virulent or attenuated strains of MG.The results showed that the detection limit of this LAMP assay was 10 fg,which was 102-fold higher than that of the conventional PCR.There was no cross reaction with other pathogens of chicken.The amplification could be finished by water bath within 1 h,and the pre-sence of MG could be detected and visualized by naked eyes.These results suggested that the LAMP assay was a simple,rapid,sensitive and specific method for the differential detection of MG in the field.