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

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.