9种蜱媒病原体xMAP 悬浮芯片高通量检测方法的建立

doi:10.11853/j.issn.1003.4692.2013.05.005

中国媒介生物学及控制杂志 ›› 2013, Vol. 24 ›› Issue (5): 397-401.DOI: 10.11853/j.issn.1003.4692.2013.05.005

9种蜱媒病原体xMAP 悬浮芯片高通量检测方法的建立

王旺, 杨宇, 王静, 赵婷婷, 孙肖红, 刘丽娟

中国检验检疫科学研究院卫生检疫研究所, 北京 100123

收稿日期:2013-04-21 出版日期:2013-10-20 发布日期:2013-10-20
通讯作者: 王静, Email: wangjing0115@126.com
作者简介:王旺(1984- ),男,主要从事传染性病原体检测技术研究。Email: ww19841016@yahoo.com.cn
基金资助:
科技部国际合作项目(2010DFA34250); 中国检验检疫科学院科研基金项目(2012JK016)

High-throughput xMAP suspension arrays for simultaneous detection of 9 tick-borne pathogens

WANG Wang, YANG Yu, WANG Jing, ZHAO Ting-ting, SUN Xiao-hong, LIU Li-juan

Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100123, China

Received:2013-04-21 Online:2013-10-20 Published:2013-10-20
Supported by:
Supported by the International Cooperation Project of Ministry of Science and Technology (No. 2010DFA34250) and the Basic Scientific Research Project of Chinese Academy of Inspection and Quarantine (No. 2012JK016)

摘要/Abstract

摘要：

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(Babesia spp.)、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。

关键词: 悬浮芯片, 蜱媒, 多重, 标签序列

Abstract:

Objective To develop xMAP suspension arrays for simultaneous detection of tick-borne pathogens including forest encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, Babesia spp., Ehrlichieae, Francisella tularensis, Coxiella burneti, Borrelia burgdorferi, and Bartonella. Methods The 9 tick-borne pathogens were divided into two groups, and a multiplex PCR system was designed with up-primers labelled with anti-TAG and down-primers labelled with biotin. The PCR products were hybridized to the magnetic microspheres coupled with x-TAG. The conjugates were tested using the Bio-plex 100 system, and the 9 tick-borne pathogens were subjected to single and multiple detections and analyses. Results The mean fluorescence intensity three times higher than that of the background was judged as positive reaction. The results of single-plex and multiplex assays for the pathogen cultures demonstrated specific positive signals, without cross reaction, and the stability, sensitivity, and specificity of the method was good. Conclusion The liquid biochip technology platform with the beads coupled with x-TAG, which can simultaneously detect 9 tick-borne pathogens, has been successfully established. This platform holds promise for detection of tick-borne pathogens.

Key words: Suspension array, Tick-borne pathogen, Simultaneous detection, x-TAG

中图分类号:

王旺, 杨宇, 王静, 赵婷婷, 孙肖红, 刘丽娟. 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.

WANG Wang, YANG Yu, WANG Jing, ZHAO Ting-ting, SUN Xiao-hong, LIU Li-juan. High-throughput xMAP suspension arrays for simultaneous detection of 9 tick-borne pathogens[J]. Chines Journal of Vector Biology and Control, 2013, 24(5): 397-401.

参考文献

[1] 冯云. 重要虫媒病毒传播媒介的研究进展
[J]. 医学动物防制, 2008,24(3):165-169.

[2] 刘琪. 蜱及蜱传病的研究进展
[J]. 安徽农业科学,2013,41(3): 1107-1109.

[3] Dunbar SA. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection
[J]. Clin Chim Acta,2006,363(1/2):71-82.

[4] 杨宇,张晓龙. 2010年中蒙边境甘其毛都口岸蜱携带病原体调查研究
[J]. 中国国境卫生检疫杂志,2011,34(5):339-342.

[5] 杨晓军,陈泽,刘敬泽. 中国蜱类的有效属和有效种
[J]. 河北师范大学学报:自然科学版,2008,32(4):529-533.

[6] Piesman J, Mather TN, Telford SR, et al. Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini
[J]. J Clin Microbiol,1986,24(3):446-447.

[7] Anderson JF, Johnson RC, Magnarelli LA, et al. Peromyscus leucopus and Microtus pennsylvanicus simultaneously infected with Borrelia burgdorferi and Babesia microti
[J]. J Clin Microbiol,1986,23(1):135-137.

[8] Stafford KC, Massung RF, Magnarelli LA, et al. Infection with agents of human granulocytic ehrlichiosis, lyme disease, and babesiosis in wild white-footed mice (Peromyscus leucopus) in Connecticut
[J]. J Clin Microbiol,1999,37(9):2887-2892.

[9] Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of coinfection with Borrelia burgdorferi, Babesia microti, and human granulocytic Ehrlichia species in residents of Wisconsin and Minnesota
[J]. J Clin Microbiol,1996,34(3):724-727.

[10] Pabbaraju K, Wong S, Tokaryk KL, et al. Comparison of the Luminex x-TAG respiratory viral panel with x-TAG respiratory viral panel fast for diagnosis of respiratory virus infections
[J]. J Clin Microbiol,2011,49(5):1738-1744.

[11] Liu J, Kibiki G, Maro V, et al. Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis
[J]. J Clin Virol,2011,50(4):308-313.

[12] Iannone MA, Consler TG, Pearce KH, et al. Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres
[J]. Cytometry,2001,44(4):326-337.

[13] 罗渊,刘伯华,杨保安. 5种虫媒病毒悬浮芯片的建立
[J]. 解放军医学杂志,2008,33(6):708-711.

[14] Taylor JD, Briley D, Nguyen Q, et al. Flow cytometric platform for high-throughput single nucleotide polymorphism analysis
[J]. Biotechniques,2001,30(3):661-666,668-669.

[15] Janse I, Bok JM, Hamidjaja RA, et al. Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays
[J]. PLoS One,2012,7(2):e31958.

[16] Jobs M, Eriksson R, Blomberg J. Quantitative and multiplex detection of pathogenic fungi using padlock probes, generic qPCR, and suspension array readout
[J]. Methods Mol Biol,2013,968: 105-118.

9种蜱媒病原体xMAP 悬浮芯片高通量检测方法的建立

High-throughput xMAP suspension arrays for simultaneous detection of 9 tick-borne pathogens

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