9种蜱媒病原体xMAP悬浮芯片高通量检测方法的建立

High throughput xMAP suspension arrays for simultaneous detection of 9 tick-borne pathogens

目的建立森林脑炎病毒(forest encephalitis,TBEV)、新疆出血热病毒(Xinjiang hemorrhagic fever,XHF)、斑点热群立克次体(spotted fever group rickettsiae,SF)、巴贝西原虫(Babesiaspp.)、埃立克体(Ehrlichieae)、土拉弗朗西斯菌(Francisella tularensis,Fr)、Q热立克次体(Coxiella burneti,Q)、伯氏疏螺旋体(Borrelia burgdorferi,Bo)、巴尔通体(Bartonella,Barton)9种蜱传病原体的悬浮芯片多重检测方法。方法将9种蜱媒病原体分为两组构建多重PCR体系,各上游引物均带有不同的anti TAG标记,下游引物标记生物素,PCR产物与偶联对应x TAG的磁性微球进行杂交,结合物用Bio plex 100液相芯片系统检测,并对9种蜱媒病原体分别进行单一、多重检测分析。结果将平均荧光强度的判定阈值定为背景对照的3倍,多批次实验均能对混合模板进行准确鉴定,未出现交叉反应,表明该方法的稳定性和特异性均较好;同时对该方法的灵敏性进行鉴定,结果表明本检测方法灵敏度良好。结论通过利用偶联有标签序列及生物素的引物对,成功建立了可同时检测9种蜱媒病原的液相芯片检测技术平台;该平台对于蜱媒病原体的检测具有较好的应用前景。

Objective To develop xMAP suspension arrays for simultaneous detection of tick-borne pathogens including forest encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, Babesia spp., Ehrlichieae, Francisella tularensis, Coxiella burneti, Borrelia burgdorferi, and Bartonella. Methods The 9 tick-borne pathogens were divided into two groups, and a multiplex PCR system was designed with up-primers labelled with anti-TAG and down-primers labelled with biotin. The PCR products were hybridized to the magnetic microspheres coupled with x-TAG. The conjugates were tested using the Bio- plex 100 system, and the 9 tick-borne pathogens were subjected to single and multiple detections and analyses. Results The mean fluorescence intensity three times higher than that of the background was judged as positive reaction. The results of single- plex and multiplex assays for the pathogen cultures demonstrated specific positive signals, without cross reaction, and the stability, sensitivity, and specificity of the method was good. Conclusion The liquid biochip technology platform with the beads coupled with x-TAG, which can simultaneously detect 9 tick-borne pathogens, has been successfully established. This platform holds promise for detection of tick-borne pathogens.