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中东呼吸综合征冠状病毒微流控芯片自动检测方法建立

刘建礼 高静 蒋鹏翀 任鲁风 张奕 焦艳丽 包阔 肖利力

刘建礼, 高静, 蒋鹏翀, 任鲁风, 张奕, 焦艳丽, 包阔, 肖利力. 中东呼吸综合征冠状病毒微流控芯片自动检测方法建立[J]. 中国公共卫生, 2023, 39(1): 92-97. doi: 10.11847/zgggws1139190
引用本文: 刘建礼, 高静, 蒋鹏翀, 任鲁风, 张奕, 焦艳丽, 包阔, 肖利力. 中东呼吸综合征冠状病毒微流控芯片自动检测方法建立[J]. 中国公共卫生, 2023, 39(1): 92-97. doi: 10.11847/zgggws1139190
LIU Jian-li, GAO Jing, JIANG Peng-chong, . Establishment of an automatic detection method for Middle East respira-tory syndrome coronavirus with an integrated microfluidic chip[J]. Chinese Journal of Public Health, 2023, 39(1): 92-97. doi: 10.11847/zgggws1139190
Citation: LIU Jian-li, GAO Jing, JIANG Peng-chong, . Establishment of an automatic detection method for Middle East respira-tory syndrome coronavirus with an integrated microfluidic chip[J]. Chinese Journal of Public Health, 2023, 39(1): 92-97. doi: 10.11847/zgggws1139190

中东呼吸综合征冠状病毒微流控芯片自动检测方法建立

doi: 10.11847/zgggws1139190
基金项目: 国家重点研发计划项目(2019YFF0302501;2021YFC2401000)
详细信息
    作者简介:

    刘建礼(1973 – ),男,山东人,主任技师,博士,主要从事传染病检测及技术研发工作

    通讯作者:

    肖利力,E-mail:bithc2016@163.com

  • 中图分类号: R 373.1

Establishment of an automatic detection method for Middle East respira-tory syndrome coronavirus with an integrated microfluidic chip

  • 摘要:   目的  建立基于微流控芯片平台的中东呼吸综合征冠状病毒(MERS-CoV)一体化自动检测方法。  方法  制备MERS-CoV假病毒颗粒阳性参考品,设计针对病毒ORF1b基因区的引物和探针,制备基于实时荧光RT-PCR全流程的一体化集成微流控芯片,实现含样品裂解、核酸提取、核酸扩增和荧光检测的全自动检测。利用芯片内预包埋的内参基因对芯片系统性能进行监测,利用MERS-CoV假病毒阳性参考品对方法的灵敏度、特异性和重复性检测评价。  结果  微流控芯片中内参基因可以稳定扩增。自动化方法可检测原始样本中100拷贝(copies)的MERS-CoV假病毒,优于常规实时荧光RT-PCR(500 copies检出限);100 copies假病毒重复测试6次CT值为(39.80 ± 0.53),变异系数为1.34 %。用自动化方法检测其他冠状病毒和人源基因均为阴性。  结论  建立的MERS-CoV微流控芯片一体化自动检测方法具有良好的稳定性、灵敏度和特异性,操作简便、快速,不受实验场地限制,可用于中东呼吸综合征现场即时检测。
  • 图  1  微流控芯片及配套检测设备实物图

    图  2  微流控芯片与常规荧光RT-PCR检测内参基因的对比

    图  3  微流控芯片与常规荧光RT-PCR的检测灵敏度比较

    图  4  MERS-CoV一体化全自动核酸检测的重复性

    表  1  MERS-CoV及内参基因的引物和探针序列

    名称序列长度基因位置
    MERS-F TTCGATGTTGAGGGTGCTCAT 21 18266~18286
    MERS-R TCACACCAGTTGAAAATCCTAATTG 25 18323~18347
    MERS-P CCCGTAATGCATGTGGCACCAATGT 25 18291~18315
    IPC-F AGTTGCAGTGTAACCGTCATGTA 22
    IPC-R TCGACGAGACTCTGCTGTTAA 22
    IPC-P CAGTAATCTGCGTCGCACGTGTGCA 20
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-05-17
  • 网络出版日期:  2023-01-09
  • 刊出日期:  2023-01-31

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