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

The emergence of the plasmid-encoded colistin-resistance gene in represents a new threat to the treatment of infection in the clinical setting. A sensitive and rapid molecular method for detection of the 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 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 gene were determined. All 20 clinically resistant isolates without the 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 -positive . During clinical screening in a major hospital in Beijing, China, seven isolates were identified as positive from the 556 isolates. In conclusion, the LAMP assay we developed was useful for detection of the gene in the clinical setting.