<p>Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of <em>mcr-1</em> to <em>mcr-5</em> in colistin-resistant bacteria</p>

Abstract

<b>Purpose:</b> The discovery of the plasmid-mediated colistin resistance genes, <i>mcr</i>, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of <i>mcr</i> genes, a convenient and reliable method to detect <i>mcr</i> genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect <i>mcr</i> genes (<i>mcr-1</i> to <i>mcr-5</i>) harbored by colistin-resistant bacteria. <b>Methods:</b> A triple-LAMP assay for <i>mcr-1, mcr-3</i>, and <i>mcr-4</i> and a double-LAMP assay for <i>mcr-2</i> and <i>mcr-5</i> were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. <b>Results:</b> The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific <i>mcr</i> genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. <b>Conclusion:</b> The multi-LAMP assay is a potentially promising method to detect <i>mcr</i> genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect <i>mcr-2</i> to <i>mcr-5</i> genes and the first time that multi-LAMP has been applied to detect <i>mcr</i> genes.