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

Abstract

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, 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 mcr genes, a convenient and reliable method to detect mcr 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 mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr 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. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr 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 mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Keywords: colistin resistance; enzyme digestion; mcr genes; multi-LAMP; rapid detection.

Figure 1

Locations and sequences of mcr genes used to design multi-LAMP primers. (A–E) The nucleotide sequences of the target strands of mcr-1 to mcr-5 genes. Right arrows indicate the original sequences and left arrows indicate the complementary sequences.

Figure 2

Sensitivity of the loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. (A–E) Visual detection of the LAMP amplification products of mcr-1 to mcr-5 genes with SYBR Green I. (F–J) Agarose gel electrophoresis was conducted for the LAMP products of mcr-1 to mcr-5 genes. (K–O) Comparative agarose gel electrophoresis analysis of products of the PCR assay and the corresponding LAMP assay. Lane M, Trans 2K plus II DNA marker; Lanes 1–7, serial 10-fold dilutions of templates from 10⁸ copies/µL to 10² copies/µL; Lane 8, negative (water).

Figure 3

Specificity of the loop-mediated isothermal amplification (LAMP) assays. (A–E) Visual detection of the LAMP amplification products with SYBR Green I. (F–J) Agarose gel electrophoresis of the LAMP products. Lane M: Trans 2K plus II DNA marker; Lanes 1–5: mcr-1 to mcr-5 genes; Lane 6: bla_KPC-2; Lane 7: bla_NDM-1; Lane 8: bla_CTX-M-9; Lane 9: negative (water).

Figure 4

Multiplex loop-mediated isothermal amplification (multi-LAMP) detection. Agarose gel electrophoresis and enzyme digestion analysis of mcr genes was performed for the multi-LAMP products on 2% agarose gel. (A) Lane M, Trans 2K plus II DNA marker; Lane 1, restriction enzyme digestion of mcr-1 multi-LAMP products, 170 bp, 115 bp respectively; Lane 2, restriction enzyme digestion of mcr-3 multi-LAMP products, 260 bp, 155 bp respectively; Lane 3, restriction enzyme digestion of mcr-4 multi-LAMP product, 270, 230, and 185 bp respectively; Lane 4, restriction enzyme digestion of mixed mcr-1, mcr-3, and mcr-4 multi-LAMP products, 260, 225, 170, 120, and 90 bp; Lane 5, negative (water). (B) Lane M, Trans 2K plus II DNA marker; Lane 1, restriction enzyme digestion of mcr-2 multi-LAMP products, 220, 175, and 140 bp respectively; Lane 2, restriction enzyme digestion of mcr-5 multi-LAMP products, 120 bp, 90 bp respectively; Lane 3, restriction enzyme digestion of mixed mcr-2 and mcr-5 multi-LAMP products, 215, 170, 140, 120, and 90 bp, respectively; Lane 4: negative (water).