新型液相芯片复合检测5种鼠传病原体方法的建立

    王旺，杨宇，王静，赵婷婷

    中国检验检疫科学研究院卫生检疫研究所，北京 100123

    摘要：目的 建立一种能同时检测5种鼠传病原体：流行性出血热病毒（Epidemichemorrhagic fever，EHF）、斑疹伤寒立克次体（Rickettsiatyphi，TY1）、鼠疫耶尔森菌（Yersinia pestis，YP）、恙虫病立克次体（Rickettsiatsutsugamushi，RST）、钩端螺旋体（Leptospira，Lep）的液相芯片多重检测方法；方法针对5种病原的全基因组序列，分别设计特异性引物，并将上游引物5’端分别偶联一些特定的核酸片段作为标签（TAG），下游引物5’端标记生物素，PCR扩增后产物与微球进行杂交，最后利用液相芯片检测系统分析结果。结果建立的方法能同时完成对5种病原体Bio-plex100 病原体及其中任何1种的检测，特异性好，灵敏度最低可达3 fg/test。结论通过利用偶联有TAG及生物素的引物对，成功建立了可同时检测5种鼠传病原的液相芯片检测方法，具有较高的灵敏度和特异性。该方法对于鼠传病原体的检测具有较好的应用前景。

    关键词：液相芯片；鼠传病原体；复合检测；标签

    中图分类号：R51　文献标识码：B

    Modern multiplexed suspension arrays forsimultaneous detection of five Rodent-borne pathogens

    WANG Wang, YANG Yu, WANG Jing, ZHAOTing-ting

    Institute of Health Quarantine, ChineseAcademy of Inspection and Quarantine, Beijing 100013,China

    Abstract:   Objective  To develop a new multiplexed X-TAG suspension arrays forsimultaneous detection of rodent-borne pathogens including Epidemichemorrhagic fever (EHF), Rickettsia typhi(TY1), Yersiniapestis(YP), Rickettsia tsutsugamushi (RST) and Leptospira(Lep).    Methods   Multi-PCR weredesigned with up-primers labeled by TAG on 5’ end and down-primerslabeled by biotin on 5’ end separately, the PCR amplicons thenhybridized to the coded magnetic microspheres coupled withanti-TAG, the conjugates were test and results were analyzed onBio-plex 100.   Results   The method could beused to detect pathogens of any of these five types of rodent-bornepathogens and the sensitivity can reach 3 fg/testlowest.   Conclusion   The liquid biochiptechnology platform with the beads coupling with TAG wasestablished which can simultaneously detect 5 types of rodent-bornepathogens, indicating a application potential for pathogenmultiplex detection.

    Key words:   Suspension array;Rodent-borne pathogens; Simultaneous detection; TAG

    《中国国境卫生检疫杂志》2013年第2期