以dotA基因为靶标的LAMP法快速检测嗜肺军团菌

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。

Rapid detection of Legionella pneumophila by LAMP approach using dotA gene as target

Objective To develop a simple and rapid method for detection of Legionella pneumophila,the major pathogen of legionellosis,with high specificity and sensitivity.Methods Loop-mediated isothermal amplification (LAMP) was applied in this study and Type ⅣB secretion system (T4BSS) of L.pneumophila was analyzed for a specific gene suitable for the LAMP template.The dotA gene was finally selected as the amplification target.For specificity and sensitivity evaluation,the genomic DNA from L.jordanis,L.maceachernii,Salmonella and different serotypes of L.pneumophila were detected and gradient concentrations of the positive template were analyzed,while the conventional PCR method was chosen as parallel tests.The approach was also applied to the detection of environmental water samples.Results In this approach,the amplification could finish within 30 minutes.The electrophoresis image of the products presented an LAMP-characteristic ladder pattern,and the fragment size of the digested products consisted with the theoretical values.The positive tubes produced visible white precipitation,and presented bright fluorescence under ultraviolet after adding ethidium bromide (EB),while the negative controls did not present the same phenomena obviously.Conclusion This method has higher specificity as well as higher sensitivity than the conventional PCR,and it has the rapid,specific,sensitive and visual advances in the detection of L.pneumophila,requiring no special equipment,and therefore,it has a potential for wider use in the field detection.