Figures & data
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: blaKPC-2; Lane 7: blaNDM-1; Lane 8: blaCTX-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).
Table 1 mcr genes and other resistant genes used in this study
Table 2 The primer sequences of multi-LAMP and PCR for mcr-1 to mcr-5 genes
Table S1 The primer sequences of LAMP for mcr-1 to mcr-5 genes