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目的建立一种简便、高效、特异、灵敏的快速检测军团菌肺炎致病原-嗜肺军团菌的方法。方法本研究分析了NCBI数据库中嗜肺军团菌IVB型分泌系统的序列,经过筛选建立了以dotA基因为靶标的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)检测法。为了验证该法的特异性,提取了约旦军团菌、马氏军团菌、沙门菌、不同血清型的嗜肺军团菌基因组DNA进行检测;为了验证该法的灵敏度,对梯度浓度的阳性模板进行了检测,并与普通PCR对比;最后还将该法实际应用于环境水样的检测。结果本法可于30 min内完成扩增;扩增产物电泳呈现LAMP特有的梯形条带,酶切产物的片段大小符合理论值;阳性产物中可见白色沉淀,加入溴乙锭后在紫外光下可见明亮的荧光,阴性产物无此现象。结论该dotA-LAMP嗜肺军团菌检测法的特异性好,灵敏度高于普通PCR,可在简易的实验环境中快速、特异、灵敏、直观地检测出嗜肺军团菌,具备现场检测及基层推广的应用潜力。
Objective To establish a simple, efficient, specific and sensitive rapid detection of Legionella pneumophila pneumophila pathogens - Legionella pneumophila method. Methods The sequence of the IVB secretion system of Legionella pneumophila in the NCBI database was analyzed in this study. Loop-mediated isothermal amplification (LAMP) was established by screening dotA gene. In order to verify the specificity of this method, Legionella pneumophila, Legionella spp., Salmonella spp. And Legionella pneumophila strains of different serogroups were extracted for genomic DNA testing. To verify the sensitivity of this method, a positive template of gradient concentration Detection, and compared with ordinary PCR; Finally, the method is also actually applied to the detection of environmental water samples. Results The method was able to complete the amplification within 30 min. The electrophoresis of PCR products showed LAMP-specific trapezoidal bands. The fragment size of the digested products corresponded with the theoretical value. The white precipitate appeared in the positive product. After addition of ethidium bromide, Visible bright fluorescence, negative products without this phenomenon. Conclusion The detection of Legionella pneumophila by dotA-LAMP is rapid, specific, sensitive and intuitive in a simple experimental environment with good specificity and sensitivity. The detection of Legionella pneumophila with on-site detection and grassroots promotion Application potential.