3种蜱传细菌性病原体多重实时荧光定量PCR法的建立

doi:10.11853/j.issn.1003.8280.2024.03.014

中国媒介生物学及控制杂志 ›› 2024, Vol. 35 ›› Issue (3): 334-338.DOI: 10.11853/j.issn.1003.8280.2024.03.014

3种蜱传细菌性病原体多重实时荧光定量PCR法的建立

胡伟超^1,2, 李晋宇^1,2, 赵宁², 刘起勇², 温红玲¹, 吴海霞²

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

收稿日期:2024-03-12 出版日期:2024-06-20 发布日期:2024-06-29
通讯作者: 吴海霞,E-mail:wuhaixia@icdc.cn;温红玲,E-mail:whling@sdu.edu.cn
作者简介:胡伟超,男,在读硕士,主要从事蜱传疾病研究,E-mail：hwcstu@163.com
基金资助:
国家科技重大专项（2017ZX10303404-006-002）；媒介生物监测与控制项目（102393220020000012）

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

HU Wei-chao^1,2, LI Jin-yu^1,2, ZHAO Ning², LIU Qi-yong², WEN Hong-ling¹, WU Hai-xia²

1. 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;
2. 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

Received:2024-03-12 Online:2024-06-20 Published:2024-06-29
Supported by:
National Science and Technology Major Project of China (No. 2017ZX10303404-006-002); Vector Surveillance and Control Project (No. 102393220020000012)

摘要/Abstract

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。

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

Abstract: 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.

Key words: Anaplasma phagocytophilum, Borrelia burgdorferi, Spotted fever group Rickettsia, Multiplex real-time fluorescence quantitative PCR

中图分类号:

R384.4
R372

胡伟超, 李晋宇, 赵宁, 刘起勇, 温红玲, 吴海霞. 3种蜱传细菌性病原体多重实时荧光定量PCR法的建立[J]. 中国媒介生物学及控制杂志, 2024, 35(3): 334-338.

HU Wei-chao, LI Jin-yu, ZHAO Ning, LIU Qi-yong, WEN Hong-ling, WU Hai-xia. Establishment of a multiplex real-time fluorescence quantitative PCR method for three tick-borne bacterial pathogens[J]. Chinese Journal of Vector Biology and Control, 2024, 35(3): 334-338.

参考文献

[1] Chen Z, Yang XJ. Systematics and taxonomy of Ixodida[M]. Beijing： Science Press, 2021： 126. （in Chinese）. 陈泽,杨晓军. 蜱的系统分类学[M]. 北京：科学出版社,2021：126.
[2] Fang LQ,Liu K,Li XL,et al. Emerging tick-borne infections in Chinese mainland：An increasing public health threat[J]. Lancet Infect Dis,2015,15（12）：1467-1479. DOI：10.1016/S1473-3099（15）00177-2.
[3] Boulanger N,Boyer P,Talagrand-Reboul E,et al. Ticks and tick-borne diseases[J]. Med Mal Infect,2019,49（2）：87-97. DOI：10.1016/j.medmal.2019.01.007.
[4] Dantas-Torres F,Chomel BB,Otranto D. Ticks and tick-borne diseases：A one health perspective[J]. Trends Parasitol,2012,28（10）：437-446. DOI：10.1016/j.pt.2012.07.003.
[5] Yu ZJ,Wang H,Wang TH,et al. Tick-borne pathogens and the vector potential of ticks in China[J]. Parasit Vectors,2015,8：24. DOI：10.1186/s13071-014-0628-x.
[6] Luan YX,Gou JM,Zhong DJ,et al. The tick-borne pathogens：An overview of China’s situation[J]. Acta Parasitol,2023,68（1）：1-20. DOI：10.1007/s11686-023-00658-1.
[7] Zhao GP,Wang YX,Fan ZW,et al. Mapping ticks and tick-borne pathogens in China[J]. Nat Commun,2021,12（1）：1075. DOI：10.1038/s41467-021-21375-1.
[8] Henningsson AJ,Hvidsten D,Kristiansen BE,et al. Detection of Anaplasma phagocytophilum in Ixodes ricinus ticks from Norway using a realtime PCR assay targeting the Anaplasma citrate synthase gene gltA[J]. BMC Microbiol,2015,15：153. DOI：10.1186/s12866-015-0486-5.
[9] Chiappa G,Cafiso A,Monza E,et al. Development of a PCR for Borrelia burgdorferi sensu lato,targeted on the groEL gene[J]. Folia Parasitol,2020,67：2020.026. DOI：10.14411/fp.2020.026.
[10] Reller ME,Dumler JS. Optimization and evaluation of a multiplex quantitative PCR assay for detection of nucleic acids in human blood samples from patients with spotted fever rickettsiosis,typhus rickettsiosis,scrub typhus,monocytic ehrlichiosis,and granulocytic anaplasmosis[J]. J Clin Microbiol,2020,58（9）：e01802-19. DOI：10.1128/JCM.01802-19.
[11] Răileanu C,Tauchmann O,Vasić A,et al. Borrelia miyamotoi and Borrelia burgdorferi （sensu lato） identification and survey of tick-borne encephalitis virus in ticks from north-eastern Germany[J]. Parasit Vectors,2020,13（1）：106. DOI：10.1186/s13071-020-3969-7.
[12] Yang XN. High-throughput sequencing and multiplex real-time PCR methods for the detection of tick-borne pathogens[D]. Beijing：Chinese Center for Disease Control and Prevention,2021. DOI：10.27511/d.cnki.gzyyy.2021.000087.（in Chinese）杨小娜. 高通量测序和多重荧光定量PCR方法检测蜱传病原菌研究[D]. 北京：中国疾病预防控制中心,2021. DOI：10.27511/d.cnki.gzyyy.2021.000087.
[13] Stańczak J,Cieniuch S,Lass A,et al. Detection and quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus ticks from urban and rural environment,northern Poland,by real-time polymerase chain reaction[J]. Exp Appl Acarol,2015,66（1）：63-81. DOI：10.1007/s10493-015-9887-2.
[14] Reller ME,Dumler JS. Development and clinical validation of a multiplex real-time quantitative PCR assay for human infection by Anaplasma phagocytophilum and Ehrlichia chaffeensis[J]. Trop Med Infect Dis,2018,3（1）：14. DOI：10.3390/tropicalmed3010014.
[15] Zhang JB,Wen BH,Chen ML,et al. Development of a quantitative real-time polymerase chain reaction assay specific for Anaplasma phagocytophila[J]. Chin J Zoonoses,2006,22（4）：289-293. DOI：10.3969/j.issn.1002-2694.2006.04.001.（in Chinese）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.
[16] Niu DS,Yang X,Chen ML,et al. Rapid detection of spotted fever group rickettsiae with real-time quantitative PCR[J]. Med J Chin PLA,2008,33（11）：1297-1299. DOI：10.3321/j.issn：0577-7402.2008.11.006.（in Chinese）牛东升,杨晓,陈梅玲,等. 实时荧光定量PCR检测斑点热立克次体的方法建立[J]. 解放军医学杂志,2008,33（11）：1297-1299. DOI：10.3321/j.issn：0577-7402.2008.11.006.
[17] Geng Z,Hou XX,Zhang L,et al. Evaluation of a new real-time PCR assay for detection of Borrelia burgdorferi in rodents[J]. Chin J Zoonoses,2015,31（9）：812-816. DOI：10.3969/j.issn.1002-2694.2015.09.006. 耿震,侯学霞,张琳,等. 一种莱姆病螺旋体real-time PCR方法的建立及其在鼠标本检测中的应用评价（英文）[J]. 中国人兽共患病学报,2015,31（9）：812-816. DOI：10.3969/j.issn.1002-2694.2015.09.006.
[18] Madison-Antenucci S,Kramer LD,Gebhardt LL,et al. Emerging tick-borne diseases[J]. Clin Microbiol Rev,2020,33（2）：e00083-18. DOI：10.1128/CMR.00083-18.
[19] Wang ZD,Wang B,Wei F,et al. A new segmented virus associated with human febrile illness in China[J]. N Engl J Med,2019,380（22）：2116-2125. DOI：10.1056/NEJMoa1805068.
[20] Lu M,Qin XC,Jiang YZ,et al. Emergence of ehrlichiosis by a new tick-borne Ehrlichia species in China[J]. Int J Infect Dis,2023,131：32-39. DOI：10.1016/j.ijid.2023.03.038.
[21] Cheng C,Ju WD,Wang YM,et al. Investigation of coinfection with Anaplasma phagocytophilum and tick-borne spotted fever group Rickettsia at Heilongjiang Port[J]. Port Health Control,2019,24（2）：53-59. DOI：10.3969/j.issn.1008-5777.2019.02. 017.（in Chinese）程成,鞠文东,王艳梅,等. 黑龙江口岸蜱携带斑点热群立克次体及嗜吞噬细胞无形体复合感染调查[J]. 口岸卫生控制,2019,24（2）：53-59. DOI：10.3969/j.issn.1008-5777.2019. 02.017.
[22] Pan YP,Yang JF,Niu QL,et al. Study on Borrelia burgdorferi sensu lato and spotted fever group Rickettsia in Ixodes persulcatus in Heilongjiang Province[J]. Chin Vet Sci,2017,47（1）：31-37. DOI：10.16656/j.issn.1673-4696.2017.01.005.（in Chinese）潘玉平,杨吉飞,牛庆丽,等. 黑龙江省全沟硬蜱携带伯氏疏螺旋体广义种和斑点热群立克次体的研究[J]. 中国兽医科学,2017,47（1）：31-37. DOI：10.16656/j.issn.1673-4696.2017. 01.005.
[23] 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.
[24] Stellrecht KA,Wilson LI,Castro AJ,et al. Automated real-time PCR detection of tickborne diseases using the panther fusion open access system[J]. Microbiol Spectr,2022,10（6）：e0280822. DOI：10.1128/spectrum.02808-22.
[25] Gaines DN,Operario DJ,Stroup S,et al. Ehrlichia and spotted fever group rickettsiae surveillance in Amblyomma americanum in Virginia through use of a novel six-plex real-time PCR assay[J]. Vector Borne Zoonotic Dis,2014,14（5）：307-316. DOI：10.1089/vbz.2013.1509.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	崔永洁, 刘益萍, 崔佳, 饶华祥, 栗冬梅, 于娟. 山西省长治市宠物犬人兽共患病病原菌及巴贝虫感染状况调查[J]. 中国媒介生物学及控制杂志, 2024, 35(2): 232-236.
[2]	向昱龙, 周敬祝, 张燕, 胡勇, 梁文琴. 贵州省少数民族自治州微小扇头蜱的宏基因组分析[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 319-325.
[3]	段立科, 侯学霞, 张琳, 包子豪, 刘泽梁, 施淇源, 周海健, 董爱英, 郝琴. 内蒙古阿尔山地区游离蜱携带伯氏疏螺旋体的检测及基因分型研究[J]. 中国媒介生物学及控制杂志, 2022, 33(5): 642-647.
[4]	高杨, 许士奇, 王丽娜, 郭文平, 刘铭. 塞罕坝自然保护区蜱媒斑点热群立克次体检测及遗传进化分析[J]. 中国媒介生物学及控制杂志, 2022, 33(2): 252-257.
[5]	李基旭, 朴文, 金光俊. 长角血蜱经卵传播斑点热群立克次体新基因型Candidatus Rickettsia longicornii的研究[J]. 中国媒介生物学及控制杂志, 2021, 32(2): 139-143.
[6]	郝琴. 莱姆病的流行现状及防制措施[J]. 中国媒介生物学及控制杂志, 2020, 31(6): 639-642.
[7]	张学潮, 朱函坪, 姚苹苹, 徐海君, 徐芳, 孙一晟, 卢杭景, 张云, 岳明, 杨章女. 伯氏疏螺旋体外膜蛋白OspC和鞭毛蛋白FlaB多克隆抗体的制备和免疫原性研究[J]. 中国媒介生物学及控制杂志, 2020, 31(5): 531-535.
[8]	张琳, 文书, 侯学霞, 李沙, 杨小娜, 陈婷, 苗广青, 符晓莹, 郝琴, 朱雄. 海南省琼中地区寄生蜱携带伯氏疏螺旋体的检测及基因分型研究[J]. 中国媒介生物学及控制杂志, 2020, 31(4): 426-429.
[9]	段兴德, 何志海, 高子厚, 蒋宝贵, 龚正达, 张云, 邵宗体, 江佳富, 孙毅, 刘洪波, 姚明国, 王帆, 杜春红. 云南省边境地区卵形硬蜱中中华基因型伯氏疏螺旋体的检出和鉴定[J]. 中国媒介生物学及控制杂志, 2019, 30(5): 519-523.
[10]	刘维俊, 肖方震, 林思娴, 韩腾伟, 周淑姮, 邓艳琴. 福建省5县（市）鼠类感染伯氏疏螺旋体的调查研究[J]. 中国媒介生物学及控制杂志, 2019, 30(3): 320-323.
[11]	王双青, 占炳东, 张建民, 曹国平, 钟建跃, 余樟有. 浙江省衢州市开化县鼠类伯氏疏螺旋体感染状况调查[J]. 中国媒介生物学及控制杂志, 2018, 29(6): 601-603.
[12]	张琳, 苗广青, 侯学霞, 李博, 郝琴. 巢式PCR和实时荧光定量PCR在莱姆病宿主动物监测中的应用评价[J]. 中国媒介生物学及控制杂志, 2018, 29(5): 425-427.
[13]	王卓, 王建伟, 于淼, 邢悦鹏, 冯立, 杨义军, 王秀红, 田圃, 吴益民. 东北地区蜱传斑点热群立克次体的分子流行病学研究[J]. 中国媒介生物学及控制杂志, 2018, 29(4): 344-347.
[14]	叶海波, 万道正, 许佳, 云小云, 张晓龙, 覃凤葵, 陈健超, 方勇, 姚李四. 广西地区部分口岸鼠传疾病病原体调查[J]. 中国媒介生物学及控制杂志, 2017, 28(4): 343-346.
[15]	高娃, 殷旭红, 郭生春, 乌兰图雅, 曹民治. 嗜吞噬细胞无形体在不同组织细胞间抗原差异研究[J]. 中国媒介生物学及控制杂志, 2017, 28(3): 255-257.