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目的建立一种检测伯氏疏螺旋体的分型多重实时荧光定量PCR方法,可对3种致病基因型伽氏疏螺旋体B.garinii、阿氏疏螺旋体B.afzelii、狭义伯氏疏螺旋体B.burgdorferi sensu stricto检出并分型。方法针对3种基因型菌株的外膜蛋白osp C基因,在保守序列设计通用引物并分别设计型特异Taqman探针,3条探针分别标记FAM、Texas Red和CY5荧光报告基团,建立和优化多重实时荧光定量PCR反应体系,检测其敏感性和特异性。结果建立的多重荧光定量PCR的最低检测值为B.garinii 40copies/μl、B.afzelii 5.17copies/μl和B.burgdorferi sensu stricto 37.8copies/μl,通过检测立克次体、土拉弗朗西斯菌无交叉反应,检测100只游离蜱与常规PCR结合测序结果分型一致,说明本方法有较好的灵敏性和特异性。结论该方法能快速检测伯氏疏螺旋体并同时分型,为莱姆病的诊断提供新方法。
OBJECTIVE: To establish a multiplex real-time PCR method for the detection of Borrelia burgdorferi, which can detect three pathogenic genotypes, B. garinii, B. afzelii, B. burgdorferi B. burgdorferi sensu stricto detected and type. Methods Based on the outer membrane protein osp C gene of three genotypes, universal primers were designed in the conserved sequence and specific Taqman probes were designed respectively. Three probes labeled FAM, Texas Red and CY5 fluorescent reporter groups, respectively, to establish and optimize Multiple real-time fluorescence quantitative PCR reaction system to detect the sensitivity and specificity. Results The detection limit of multiplex fluorescence quantitative PCR was B.garinii 40copies / μl, B.afzelii 5.17copies / μl and B.burgdorferi sensu stricto 37.8copies / μl, and there was no cross between Rickettsia The reaction and detection of 100 free ticks were consistent with the sequencing results of conventional PCR, indicating that the method has good sensitivity and specificity. Conclusion This method can detect Borrelia burgdorferi rapidly and simultaneously and provide a new method for the diagnosis of Lyme disease.