7种鼠传病原体多重PCR检测方法的建立与应用

doi:10.11853/j.issn.1003.8280.2019.05.002

中国媒介生物学及控制杂志 ›› 2019, Vol. 30 ›› Issue (5): 487-493.DOI: 10.11853/j.issn.1003.8280.2019.05.002

7种鼠传病原体多重PCR检测方法的建立与应用

康央^1,2, 宋秀平¹, 张燕君², 刘起勇¹, 栗冬梅¹

1 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, 传染病预防控制国家重点实验室, 感染性疾病诊治协同创新中心, 北京 102206;
2 山东大学生命科学学院, 山东青岛 266237

收稿日期:2019-06-20 出版日期:2019-10-20 发布日期:2019-10-20
通讯作者: 栗冬梅,Email:lidongmei@icdc.cn;刘起勇,Email:liuqiyong@icdc.cn
作者简介:康央,女,硕士,从事鼠传病原体检测工作,Email:kangyangky@163.com
基金资助:
国家科技重大专项（2017ZX10303404）

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

KANG Yang^1,2, SONG Xiu-ping¹, ZHANG Yan-jun², LIU Qi-yong¹, LI Dong-mei¹

1 State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
2 School of Life Sciences Shandong University

Received:2019-06-20 Online:2019-10-20 Published:2019-10-20
Supported by:
Supported by the National Science and Technology Major Project of China (No. 2017ZX10303404)

摘要/Abstract

摘要： 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。

关键词: 鼠传病原体, 多重PCR, 毛细管电泳, 温度转换PCR, 人兽共患病

Abstract: 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.

Key words: Rodent-borne pathogen, Multiplex PCR, Capillary electrophoresis, Temperature-switch PCR, Zoonosis

中图分类号:

康央, 宋秀平, 张燕君, 刘起勇, 栗冬梅. 7种鼠传病原体多重PCR检测方法的建立与应用[J]. 中国媒介生物学及控制杂志, 2019, 30(5): 487-493.

KANG Yang, SONG Xiu-ping, ZHANG Yan-jun, LIU Qi-yong, LI Dong-mei. Establishment and application of a multiplex PCR detection method for seven rodent-borne pathogens[J]. Chines Journal of Vector Biology and Control, 2019, 30(5): 487-493.

参考文献

[1] 余向华,张孝和,倪庆翔,等. 鼠传疾病流行病学研究进展[J]. 中国媒介生物学及控制杂志,2015,26(6):634-636,IV. DOI:10.11853/j.issn.1003.4692.2015.06.026.
[2] Bordes F,Blasdell K,Morand S. Transmission ecology of rodent-borne diseases:new frontiers[J]. Integr Zool,2015,10(5):424-435. DOI:10.1111/1749-4877.12149.
[3] Liu QY,Xu WB,Lu S,et al. Landscape of emerging and re-emerging infectious diseases in China:impact of ecology,climate,and behavior[J]. Front Med,2018,12(1):3-22. DOI:10.1007/s11684-017-0605-9.
[4] Tabone T,Mather DE,Hayden MJ. Temperature switch PCR (TSP):robust assay design for reliable amplification and genotyping of SNPs[J]. BMC Genomics,2009,10:580. DOI:10.1186/1471-2164-10-580.
[5] Fulop M,Leslie D,Titball R. A rapid,highly sensitive method for the detection of Francisella tularensis in clinical samples using the polymerase chain reaction[J]. Am J Trop Med Hyg,1996,54(4):364-366. DOI:10.4269/ajtmh.1996.54.364.
[6] 史永林,王誓闻,胡万富,等. 安徽地区家畜嗜吞噬细胞无形体16S rRNA基因检测和序列分析[J]. 安徽预防医学杂志,2013,19(5):329-330,338.
[7] Brenner DJ,O'Connor SP,Winkler HH,et al. Proposals to unify the genera Bartonella and Rochalimaea,with descriptions of Bartonella quintana comb. nov.,Bartonella vinsonii comb. nov.,Bartonella henselae comb. nov.,and Bartonella elizabethae comb. nov.,and to remove the family Bartonellaceae from the order Rickettsiales[J]. Int J Syst Bacteriol,1993,43(4):777-786. DOI:10.1099/00207713-43-4-777.
[8] 中国疾病预防控制中心. 恙虫病预防控制技术指南[Z]. 北京:中疾控疾发[2009] 1号,2009.
[9] 徐建国,阚飙,张建中,等. 现场细菌学[M]. 北京:科学出版社,2011:556-627.
[10] Diaz MH,Bai Y,Malania L,et al. Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes[J]. J Clin Microbiol,2012,50(5):1645-1649. DOI:10.1128/JCM.06621-11.
[11] 付秀萍,贺金荣,张景山,等. TaqMan-MGB探针实时荧光定量PCR检测恙虫病东方体方法的建立[J]. 中国媒介生物学及控制杂志,2012,23(2):108-110.
[12] 杨晓,陈梅玲,温博海,等. 实时荧光定量PCR检测莫氏立克次体[J]. 解放军医学杂志,2007,32(10):1054-1056. DOI:10.3321/j.issn:0577-7402.2007.10.016.
[13] 张晶波,温博海,陈梅玲,等. 荧光定量PCR检测嗜吞噬细胞无形体[J]. 中国人兽共患病学报,2006,22(4):289-293. DOI:10.3969/j.issn.1002-2694.2006.04.001.
[14] 史清海,曲识,周冬生,等. 应用TaqMan荧光定量PCR检测土拉弗朗西斯菌[J]. 生物技术通讯,2009,20(6):806-809.
[15] 张翠彩,李秀文,聂一新,等. 致病性钩端螺旋体TaqMan Real-time PCR检测技术的建立及其应用[J]. 中华流行病学杂志,2011,32(10):1018-1021. DOI:10.3760/cma.j.issn.0254-6450.2011.10.015.
[16] 亚红祥,赫兢,张丽娟,等. 贝氏柯克斯体实时荧光定量PCR方法的建立及对云南鼠标本检测[J]. 传染病信息,2009,22(6):345-347,350. DOI:10.3969/j.issn.1007-8134.2009.06.007.
[17] Tokarz R,Tagliafierro T,Cucura DM,et al. Detection of Anaplasma phagocytophilum,Babesia microti,Borrelia burgdorferi,Borrelia miyamotoi,and Powassan virus in ticks by a multiplex real-time reverse transcription-PCR assay[J]. mSphere,2017,2(2):e00151-17. DOI:10.1128/mSphere. 00151-17.
[18] Melničáková J,Derdáková M,Barák I. A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes[J]. Parasit Vectors,2013,6:269. DOI:10.1186/1756-3305-6-269.
[19] 王旺,杨宇,王静,等. 9种蜱媒病原体xMAP悬浮芯片高通量检测方法的建立[J]. 中国媒介生物学及控制杂志,2013,24(5):397-401. DOI:10.11853/j.issn.1003.4692.2013.05.005.
[20] 李瑾. 基于两种毛细管电泳系统检测呼吸道病毒的多重PCR技术的建立及应用[D]. 北京:中国疾病预防控制中心,2012.
[21] 李龙建,曹殿起,刘英,等. 北京市门头沟区小型兽类群落构成及鼠疫监测[J]. 中国媒介生物学及控制杂志,2013,24(3):267-269.

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

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