Sensitive and Rapid Detection of the Plasmid-Encoded Colistin-Resistance Gene mcr-1 in Enterobacteriaceae Isolates by Loop-Mediated Isothermal Amplification

Abstract

The emergence of the plasmid-encoded colistin-resistance gene <i>mcr-1</i> in <i>Enterobacteriaceae</i> represents a new threat to the treatment of infection in the clinical setting. A sensitive and rapid molecular method for detection of the <i>mcr-1</i> gene in clinical isolates is needed to control the spread of this gene. In this study, we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the <i>mcr-1</i> gene. This assay was applied to cultured bacteria and spiked human stools. Real-time monitoring of turbidity and chromogenic visualization were used to assess the reaction results. The specificity and sensitivity of the primers in the LAMP reactions for detection of the <i>mcr-1</i> gene were determined. All 20 clinically resistant isolates without the <i>mcr-1</i> gene tested negative, indicating the high specificity of the LAMP primers. The sensitivity of LAMP, with a detection limit of 0.2 pg/μL DNA, was 10-fold greater than that of polymerase chain reaction (PCR). The assay was also conclusive when applied to human stools spiked with <i>mcr-1</i>-positive <i>Escherichia coli</i>. During clinical screening in a major hospital in Beijing, China, seven isolates were identified as positive from the 556 <i>Enterobacteriaceae</i> isolates. In conclusion, the LAMP assay we developed was useful for detection of the <i>mcr-1</i> gene in the clinical setting.