检验医学 ›› 2016, Vol. 31 ›› Issue (8): 688-693.DOI: 10.3969/j.issn.1673-8640.2016.08.013

• 技术研究与评价·论著 • 上一篇    下一篇

NDM-1阳性细菌实时荧光LAMP方法的建立及应用

陈斌, 杨银梅, 钟志敏, 雷秀霞, 刘大渔, 徐邦牢   

  1. 广州医科大学附属广州市第一人民医院检验科,广东 广州 510180
  • 收稿日期:2015-11-24 出版日期:2016-08-31 发布日期:2016-09-01
  • 作者简介:null

    作者简介:陈 斌,男,1983年生,主管技师,主要从事临床分子诊断研究。

    通讯作者:徐邦牢,联系电话:020-81048082。

  • 基金资助:
    国家自然科学基金项目(81201163);广东省医学科研基金项目(B2014341);广东省自然科学基金项目(2014A030313677)

Establishment and application of real-time fluorescence LAMP for the detection of NDM-1-positive bacteria

CHEN Bin, YANG Yinmei, ZHONG Zhimin, LEI Xiuxia, LIU Dayu, XU Banglao   

  1. Department of Clinical Laboratory,Guangzhou First People's Hospital,Guangzhou Medical University,Guangzhou 510180,Guangdong,China
  • Received:2015-11-24 Online:2016-08-31 Published:2016-09-01

摘要:

目的 建立一种实时荧光环介导等温扩增(LAMP)方法快速检测新德里金属β-内酰胺酶1 (NDM-1)阳性细菌。方法 根据NDM-1 基因序列设计4套引物并进行优化,对LAMP反应中的荧光染料SYTO-9浓度进行优化,同时考察该方法的检出限和特异性。利用本方法检测72例临床分离的多重耐药鲍曼不动杆菌。结果 本研究建立的实时荧光LAMP方法,其荧光染料SYTO-9的最佳浓度为4 μmol/L;63 ℃恒温扩增35 min可检出101拷贝/μL的标准阳性模板;特异性较好;对72例临床分离的多重耐药鲍曼不动杆菌检出的阳性率为16.7%(12/72),12株NDM-1阳性细菌经测序验证,准确率为100%。结论 本研究建立的检测NDM-1阳性细菌的实时荧光LAMP方法具有快速、操作简便、特异性好、敏感性高、准确、可靠等优点,可以直观、实时地观察反应的进行情况,适合临床NDM-1阳性细菌的快速检测。

关键词: 实时荧光环介导等温扩增, 新德里金属β-内酰胺酶1细菌, 快速检测

Abstract:

Objective To establish a real-time fluorescence loop-mediated isothermal amplification(LAMP)for the detection of New Delhi metallo-beta-lactamase-1(NDM-1)-positive bacteria. Methods According to the sequence of NDM-1 gene,4 primers and fluorescent dye concentration of SYTO-9 were designed and optimized,and the limit of detection and specificity were also evaluated. A total of 72 isolates of multidrug-resistant Acinetobacter baumannii were detected. Results The optimal fluorescent dye concentration of the established real-time fluorescence LAMP was 4 μmol/L. The limit of detection was 101 copies/μL for 35 min at 63 ℃. The specificity was good. A total of 72 isolates of multidrug-resistant Acinetobacter baumannii were detected,and the positive rate was 16.7% (12/72). The 12 isolates were further confirmed by sequencing,and the accuracy was 100%. Conclusions The established real-time fluorescence LAMP for the detection of NDM-1-positive bacteria is rapid,simple,specific,sensitive,accurate and reliable,and it could be used for the routine detection of NDM-1-positive bacteria.

Key words: Real-time fluorescence loop-mediated isothermal amplification, New Delhi metallo-beta-lactamase-1 bacteria, Rapid detection

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