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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 establish forest encephalitis (TBEV), Xinjiang hemorrhagic fever (XHF), spotted fever group rickettsiae (SF), Babesiaspp. Ehrlichieae, Francisella tularensis (Fr), Coxiella burneti (Q), Borrelia burgdorferi, Bo, Bartonella (Barton) Multiplex Detection of 9 Tickborne Pathogens by Suspension Chips. Methods Nine species of tick-borne pathogens were divided into two groups to construct a multiplex PCR system. Each upstream primer contained different anti-TAG markers. The downstream primers labeled biotin. The PCR products were hybridized with the magnetic microspheres conjugated to x TAG. Bio plex 100 liquid chip system was used to detect the nine tick pathogens, respectively, single and multiple detection analysis. Results The threshold value of average fluorescence intensity was set as 3 times of the background control. The results showed that the mixed template was accurately identified by multiple batches of experiments without cross-reaction, indicating that the method was stable and specific. The sensitivity of the method was identified, the results show that the sensitivity of the detection method is good. Conclusion The liquid chromatography chip detection technology platform for simultaneous detection of nine tick pathogens has been established successfully by using primer pairs coupled with tag sequences and biotin. The platform has good application prospects for the detection of tick pathogens.