高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

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

    通信作者:

    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
  • [1] 董家潇, 李修政, 张忠伟, 等. SARS - COV和MERS - COV以及SARS-COV-2的研究进展[J]. 中国病毒病杂志, 2021, 11(5): 381 – 387. doi: 10.16505/j.2095-0136.2021.0050
    [2] World Health Organization. MERS situation update, March 2022[EB/OL]. (2022 – 02 – 20). http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html.
    [3] 陆柔剑, 武婕, 牛培华, 等. 中国首例输入性中东呼吸综合征病例实验室确诊方法比较[J]. 中华实验和临床病毒学杂志, 2015, 29(3): 193 – 195. doi: 10.3760/cma.j.issn.1003-9279.2015.03.001
    [4] 于海江, 于洋洋, 于栋, 等. SARS-CoV-2、SARS-CoV-1及MERS-CoV的特征比较分析[J]. 基因组学与应用生物学, 2020, 39(9): 4400 – 4404. doi: 10.13417/j.gab.039.004400
    [5] 王晓欢, 严延生, 张智芳, 等. 中东呼吸综合征[J]. 中国人兽共患病学报, 2020, 36(6): 496 – 502.
    [6] World Health Organization. Laboratory testing for middle east respiratory syndrome coronavirus-revised, interim guidance[EB/OL]. (2018 – 01 – 15). https://www.who.int/publications/i/item/10665-259952.
    [7] Taki K, Yokota I, Fukumoto T, et al. SARS - CoV - 2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction[J]. Journal of Infection and Chemotherapy, 2021, 27(2): 410 – 412. doi: 10.1016/j.jiac.2020.10.029
    [8] Wei S, Kohl E, Djandji A, et al. Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction[J]. Scientific Reports, 2021, 11(1): 2402.
    [9] Pearson JD, Trcka D, Lu SY, et al. Comparison of SARS - CoV - 2 indirect and direct RT - qPCR detection methods[J]. Virology Journal, 2021, 18(1): 99. doi: 10.1186/s12985-021-01574-4
    [10] Beltrán-Pavez C, Alonso-Palomares LA, Valiente-Echeverría F, et al. Accuracy of a RT - qPCR SARS - CoV - 2 detection assay without prior RNA extraction[J]. Journal of Virological Methods, 2021, 287: 113969. doi: 10.1016/j.jviromet.2020.113969
    [11] Shabani E, Dowlatshahi S, Abdekhodaie MJ. Laboratory detec-tion methods for the human coronaviruses[J]. European Journal of Clinical Microbiology and Infectious Diseases, 2021, 40(2): 225 – 246. doi: 10.1007/s10096-020-04001-8
    [12] Huang P, Wang HL, Cao ZG, et al. A rapid and specific assay for the detection of MERS - CoV[J]. Frontiers in Microbiology, 2018, 9: 1101. doi: 10.3389/fmicb.2018.01101
    [13] Shahrajabian MH, Sun WL, Cheng Q. Different methods for molecular and rapid detection of human novel coronavirus[J]. Current Pharmaceutical Design, 2021, 27(25): 2893 – 2903. doi: 10.2174/1381612827666210604114411
    [14] Ye X, Li Y, Wang LJ, et al. All-in-one microfluidic nucleic acid diagnosis system for multiplex detection of sexually transmitted pathogens directly from genitourinary secretions[J]. Talanta, 2021, 221: 121462. doi: 10.1016/j.talanta.2020.121462
    [15] Liu Q, Zhang XL, Chen LP, et al. A sample - to - answer labdisc platform integrated novel membrane - resistance valves for detection of highly pathogenic avian influenza viruses[J]. Sensors and Actuators B: Chemical, 2018, 270: 371 – 381. doi: 10.1016/j.snb.2018.05.044
    [16] Chen JG, Xu YC, Yan H, et al. Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recom-binase polymerase amplification[J]. Lab on a Chip, 2018, 18(16): 2441 – 2452. doi: 10.1039/C8LC00399H
    [17] Kim MN, Ko YJ, Seong MW, et al. Analytical and clinical validation of six commercial middle East respiratory syndrome coronavirus RNA detection kits based on real - time reverse - transcription PCR[J]. Annals of Laboratory Medicine, 2016, 36(5): 450 – 456. doi: 10.3343/alm.2016.36.5.450
    [18] 迟航, 郑学星, 盖微微, 等. 中东呼吸综合征冠状病毒实时荧光定量PCR检测方法的建立[J]. 中国病原生物学杂志, 2014, 9(8): 673 – 677. doi: 10.13350/j.cjpb.140801
  • 加载中
图(4) / 表(1)
计量
  • 文章访问数:  315
  • HTML全文浏览量:  224
  • PDF下载量:  62
  • 被引次数: 0
出版历程
  • 接收日期:  2020-05-17
  • 网络出版日期:  2023-01-09
  • 刊出日期:  2023-01-31

目录

    /

    返回文章
    返回