NDM-1阳性细菌实时荧光LAMP方法的建立及应用  

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

作　　者：陈斌[1] 杨银梅[1] 钟志敏[1] 雷秀霞[1] 刘大渔[1] 徐邦牢[1] 

机构地区：[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

出　　处：《检验医学》2016年第8期688-693,共6页Laboratory Medicine

基　　金：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)

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4μmol/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。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.

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 

分 类 号：R446.5[医药卫生—诊断学]