Rapid and Sensitive Detection of the Colistin Resistance Gene mcr-3 by Loop-Mediated Isothermal Amplification and Visual Inspection

Abstract

Objective: The distribution of colistin resistance in mcr-carrying bacteria poses a threat to global public health. In particular, the newly identified mcr-3 allele has spread globally, especially in China, second only to mcr-1. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid, sensitive, and visual detection of the presence of the mcr-3 gene. Materials and Methods: A total of 13 clinical bacterial strains and 11 negative strains were used in this study. We designed LAMP Primers, optimized reaction conditions, used three different methods to detect LAMP amplification products: (1) agarose gel electrophoresis, (2) LAMP-hydroxy naphthol blue (HNB) detection, (3) LAMP-SYBR Green I (LAMP-SGI) visual inspection, and evaluated its specificity and sensitivity. Results: The amplification reaction was completed in 1 hr at 62°C under isothermal conditions. The final optimized mixtures contained 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, 1.2 μL HNB, and 0.5 μL SYBR Green I as additives to the initial reaction mixture. LAMP detection, including two visual methods, LAMP-HNB and LAMP-SGI, of mcr-3 possessed the same specificity and a 10-fold higher sensitivity compared with a conventional polymerase chain reaction assay using the same samples. Conclusion: We successfully established an mcr-3 LAMP detection with portability and rapidity of the reaction by the easily distinguishable color changes in the reaction tubes. This visual LAMP assay for mcr-3 detection was simple, time saving, and economical, especially suited to field laboratories.