8种重大烈性传染病病毒液相芯片检测方法的建立

   欧青叶1，顾大勇2，胡春凌3，李永进4，史蕾2，刘春晓2，赵纯中2，徐云庆2，张妮奇2

    1.珠海市结核病防治所，广东 珠海 519000；2.深圳国际旅行卫生保健中心；

    3.深圳博睿祥晖生物技术有限公司；4.深圳博尔美生物科技有限公司；

    摘要：目的建立基于多重PCR技术结合液相芯片技术同时检测汉坦病毒（HTV）、裂谷热病毒（RVFV）、黄热病毒（YFV）、西尼罗病毒（WNV）、克里米亚-刚果出血热病毒（CCHFV）、拉沙热[下同]病毒（LFV）、埃博拉病毒（EBV）和马尔堡病毒（MBV）的方法。方法建立8种病毒同时扩增的多重PCR反应体系，分别用各种病毒特异的核酸探针偶联不同编码的微球，将获得的PCR产物与偶联核酸探针的微球混合物进行杂交，建立液相芯片检测方法，并对建立的液相芯片检测方法进行灵敏度及特异性检测评价。结果建立的8种重大烈性传染病病毒的液相芯片筛查方法具有较高的特异性和敏感性，能对8种病毒进行特异的检测。灵敏性实验结果表明，RVFV为10ng/PCR、WNV为1 ng/PCR、EBV为10 ng/PCR、CCHFV为10 pg/PCR、MBV为1ng/PCR、HTV为100 pg/PCR、LFV为1 ng/PCR、YFV为10 pg/PCR。结论本研究建立的病毒液相芯片筛查方法能快速、敏感、特异地同时检测8种重大烈性传染病病毒，对口岸入境人员是否携带重大烈性传染病病毒的快速筛查具有广阔的应用前景。

    关键词：烈性传染病病毒；多重PCR；液相芯片；筛查

    中图分类号：R183；R34　文献标识码：B

    Development of a luminex liquid chiptechnology for  detection of eight

    kinds of deadly infectious virus

    'OU Qing-ye*，GU Da-yong，HU Chun-ling，LIYong-jin，SHI Lei，LIU Chun-xiao，

    ZHAO CHUN-zhong，XU Yun-qing，ZHANG Ni-qi

    *ZhuHai City Tuberculosis Control Center,ZhuHai,Guangdong，519060, China

    Abstract:   Objective  To establish a Luminex assay coupled with multiplex PCR for thesimultaneous detection of Hantaan virus(HTV), Rift Valley FeverVirus(RVFV), Yellow fever virus(YFV), West Nile Virus(WNV),Crimean-Congo hemorrhagic fever virus(CCHFV ), Lassa fevervirus(LFV), Ebola virus(EBV) and Marburg virus(MBV). Methods   A luminex-based assay for simultaneouslydetection of eight foreign deadly infections virus was used,including multiplex PCR, virus-specific nucleic probe coupling withmicrospheres of different codes, and hybridization. The sensitivityand specificity of the method were evaluated by plasmid DNA whichwere synthetic.  Results   The screening method hasa high specificity and sentsitivity. The minimum detection limitfor CCHFV and YFV was 10pg/PCR; the minimum detection limit for HTVwas 10 pg/PCR; the minimum detection limit for WNV, MBV and LFV was1 ng/PCR; the minimum detection limit for RVFV and EBV was 10ng/PCR.  Conclusion   The method gives a rapid,accurate, sensitive detection of eight foreign deadly infectionsvirus in the same tube, indicating a application potential for thescreening of the foreign deadly infections virus that are carriedby port entry personnel.

    Key words:   Deadly InfectiousVirus；Multiplex PCR；Luminex assay；Screening

    《中国国境卫生检疫杂志》2012年8月刊