7种鼠传病原体多重PCR检测方法的建立与应用

Establishment and application of a multiplex PCR detection method for seven rodent-borne pathogens

目的应用QIAxcel毛细管电泳系统建立一种能够同时检测7种鼠传病原体的多重PCR检测方法。方法应用微生物数据分析云平台(https://analysis.mypathogen.org/)中'special marker gene'工作流,筛选恙虫病东方体、嗜吞噬细胞无形体、莫氏立克次体、土拉弗朗西斯菌、贝氏柯克斯体、问号钩端螺旋体和巴尔通体特异基因,据此设计引物。根据温度转换PCR原理构建特异性嵌合引物,建立多重PCR反应体系,应用QIAxcel毛细管电泳系统对扩增产物进行检测,评价该体系灵敏度、特异性和可重复性。通过对模拟样品和野外样品进行检测,评价该方法对样品的检测能力。结果特异性检测显示,每种病原体扩增均只出现单一目的条带,无交叉反应;多引物单模板灵敏度检测限为11~76拷贝/μl,多引物多模板灵敏度检测限为20~200拷贝/μl。经样品检测显示,多重PCR法检测能力与单重实时荧光定量PCR法相当,优于普通单重PCR法。结论运用QIAxcel毛细管电泳系统成功建立鼠传病原体多重PCR检测方法,可同时高效、快速检出7种病原体,为相关鼠传疾病的诊断、监测和流行病学调查提供有效手段。

Objective To establish a multiplex PCR detection method for simultaneous detection of seven rodent-borne pathogens based on the QIAxcel capillary electrophoresis system.Methods The'special marker gene'workflow in Microbial Data Analysis Cloud Platform(https://analysis.mypathogen.org/)was used to screen out the specific genes of Orientia tsutsugamushi,Anaplasma phagocytophilum,Rickettsia typhi,Francisella tularensis,Coxiella burnetii,Leptospira interrogans,and Bartonella,and specific primers were designed based on such results.Specific chimeric primers were constructed based on temperature-switch PCR,and a multiplex PCR method was established.The QIAxcel capillary electrophoresis system was used for the detection of amplification products,and the sensitivity,specificity,and repeatability of this method were evaluated.Simulated samples and field samples were tested to evaluate the detection capability of this method.Results Specificity tests showed that each pathogen had a single target band without cross reaction.The sensitivity detection limit of multi-primer single template was within the range of 11-76 copies/μl,and the sensitivity detection limit of multi-primer multiple templates was within the range of 20-200 copies/μl.Sample detection showed that the multiplex PCR detection method was comparable to the single quantitative real-time PCR method and was better than the simplex PCR method.Conclusion A multiplex PCR detection method is successfully established based on the QIAxcel capillary electrophoresis system,and this highly efficient and rapid method can detect seven rodent-borne pathogens simultaneously,which provides an effective mean for the diagnosis,monitoring,and epidemiological investigation of rodent-borne diseases.