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