3种蜱传细菌性病原体多重实时荧光定量PCR法的建立  

Establishment of a multiplex real-time fluorescence quantitative PCR method for three tick-borne bacterial pathogens

作　　者：胡伟超 李晋宇 赵宁 刘起勇[2] 温红玲 吴海霞[2] HU Wei-chao;LI Jin-yu;ZHAO Ning;LIU Qi-yong;WEN Hong-ling;WU Hai-xia(Department of Microbiological Laboratory Technology,Key Laboratory of Prevention and Control of Emerging Infectious Diseases and Biological Safety in Universities of Shandong,Department of Vector Control,School of Public Health,Cheeloo College Medicine,Shandong University,Jinan,Shandong 250012,China;Department of Vector Biology and Control,National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,WHO Collaborating Centre for Vector Surveillance and Management,Beijing 102206,China)

机构地区：[1]山东大学齐鲁医学院公共卫生学院微生物检验学系,新发突发传染病防控与生物安全山东省高等学校重点实验室,媒介生物控制学系,山东济南250012 [2]传染病溯源预警与智能决策全国重点实验室,中国疾病预防控制中心传染病预防控制所媒介生物控制室,世界卫生组织媒介生物监测与管理合作中心,北京102206

出　　处：《中国媒介生物学及控制杂志》2024年第3期334-338,共5页Chinese Journal of Vector Biology and Control

基　　金：国家科技重大专项(2017ZX10303404-006-002);媒介生物监测与控制项目(102393220020000012)。

摘　　要：目的建立一种多重实时荧光定量PCR法,同时检测常见蜱传病原体嗜吞噬细胞无形体、伯氏疏螺旋体及斑点热群立克次体(SFGR)。方法针对各病原体的保守区序列设计特异性引物及TaqMan探针,建立并优化多重实时荧光定量PCR反应体系,评价方法的特异性、灵敏度、重复性及对蜱样本的检测准确性。结果建立的多重实时荧光定量PCR法检测嗜吞噬细胞无形体、伯氏疏螺旋体、SFGR,与大肠埃希菌、问号钩端螺旋体、布鲁氏菌等病原体间无交叉反应,特异性良好;灵敏度均达到102拷贝/μl,重复性变异系数均<2.00%;蜱样本检测中,多重实时荧光定量PCR法与单重实时荧光定量PCR法检测结果一致性为100%。结论研究建立的3种蜱传细菌性病原体多重实时荧光定量PCR法特异性好、灵敏度高,为蜱传病原体的检测提供了技术手段。Objective To establish a multiplex real-time fluorescence quantitative PCR(qPCR)method for simultaneous detection of the common tick-borne pathogens Anaplasma phagocytophilum,Borrelia burgdorferi,and spotted fever group Rickettsia(SFGR).Methods Specific primers and TaqMan probes were designed against the conserved regions of pathogens,and a multiplex qPCR system was established and optimized.The specificity,sensitivity,repeatability,and accuracy for tick samples were evaluated.Results There were no cross reactivity with Escherichia coli,Leptospira interrogans,Brucella sp.in the detection of A.phagocytophilum,B.burgdorferi,and SFGR by the established multiplex qPCR method and showed high specificity.The sensitivity reached the order of 102 copies/μl,and the coefficient of variation for repeatability was less than 2.00%.In tick sample detection,the results of multiplex qPCR method were consistent with the singleplex qPCR method,with 100%consistency.Conclusion The established multiplex qPCR method for three tick-borne bacterial pathogens has high specificity and sensitivity,and can be used as a technical means for the detection of common tick-borne bacterial pathogens.

关 键 词：嗜吞噬细胞无形体 伯氏疏螺旋体 斑点热群立克次体 多重实时荧光定量PCR法 

分 类 号：R384.4[医药卫生—医学寄生虫学] R372[医药卫生—基础医学]