中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2013, Vol. 35 ›› Issue (1): 24-28.doi: 10.3881/j.issn.1000-503X.2013.01.005

• 论著 • 上一篇    下一篇

无需逆转录的高通量多重呼吸道病毒RNA检测技术的建立

杨丹丽1,田晓怡1,石伟先2,郑直1   

  1. 1中国医学科学院 北京协和医学院 基础医学研究所生物化学与分子生物学系,北京 1000052北京市疾病预防控制中心传染病地方病控制所,北京 100013
  • 收稿日期:2012-08-15 出版日期:2013-03-07 发布日期:2013-03-07
  • 通讯作者: 郑 直 电话:010-69156961,电子邮件:zhizheng10@yahoo.com E-mail:zhizheng10@yahoo.com
  • 基金资助:
    国家自然科学基金(30872412)

Establishment of a High-throughput Respiratory Viruse DetectionTechnology without RNA Purification and Reverse Transcription

YANG Dan-li1, TIAN Xiao-yi1, SHI Wei-xian2, ZHENG Zhi1   

  1. 1Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China2Insititute for Disease Control, Beijing Municipal Centers for Disease Control and Prevention, Beijing 100013, China
  • Received:2012-08-15 Online:2013-03-07 Published:2013-03-07
  • Supported by:
    Supported by the National Natural Sciences Foundation of China (30872412)

摘要: 目的 建立一种无需逆转录等复杂样品前处理步骤,只需简单样品裂解便可同时检测多种呼吸道病毒RNA的高通量核酸检测系统,为大规模流行病学监测提供新的技术手段。方法 直接以裂解液中病毒RNA作为检测靶标,利用夹层杂交信号放大技术,结合以Luminex微球为基础的多指标同步分析(xMAP)多重检测平台,对一个样品同时检测A和B型流感病毒、A和B型呼吸道合胞病毒、1、2和3型副流感病毒,及偏肺病毒等8种病毒的靶标RNA,可一次实验并行检测96个样品。对该高通量检测系统进行了特异性、灵敏度评价。结果 该检测方法可以特异性区分8种病毒的RNA靶标分子,检测下限均低于或等于4695个RNA拷贝。结论 初步建立了可靠、灵敏、操作简便、高通量的多种呼吸道病毒RNA并行检测方法,为进一步实现在临床呼吸道样品中高通量检测呼吸道病毒提供了技术基础。   

关键词: 呼吸道病毒, 体外转录RNA, 多指标同步分析技术, 信号放大

Abstract: Objective To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring. Methods We used high-throughput microsphere-based flexible multi-analyte profiling techndogy (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated. Results There was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies. Conclusion We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

Key words: respiratory viruses, in vitro-transcribed RNA, flexible multi-analyte profiling technology, signal amplification

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