Fast dissemination of the mobilized colistin resistance (
mcr
) gene
mcr-1
in
Enterobacteriaceae
causes a huge threat to the treatment of severe infection. In the current report, a multiple cross displacement amplification (MCDA) coupled with the detection of amplified products by gold nanoparticles-based lateral flow biosensor (LFB) assay (MCDA-LFB) was established to identify the
mcr-1
gene with simpleness, rapidity, specificity, and sensitivity. The MCDA-LFB assay was performed at a isothermal temperature (63°C) for only 30 min during the amplification stage, and the reaction products were directly identified by using LFB which obtained the result within 2 min. The entire process of experiments, from templates extraction to result judging, was accomplished in <60 min. For the analytical specificity of this method, all of the 16
mcr-1
-producing strains were positive, and all of the non-
mcr-1
isolates produced the negative results. The sensitivity of
mcr-1
-MCDA-LFB assay was as little as 600 fg of plasmid DNA per reaction in pure culture, and approximately 4.5 × 10
3
CFU/mL (~4.5 CFU/reaction) in spiked fecal samples. Therefore, this technique established in the present study is suitable for the surveillance of
mcr-1
gene in clinic and livestock industry.
22Fast dissemination of the mobilized colistin resistance (mcr) gene mcr-1 in 23 Enterobacteriaceae causes a huge threat to the treatment of severe infection. In the current 24 report, a multiple cross displacement amplification (MCDA) coupled with the detection of 25 amplified products by gold nanoparticles-based lateral flow biosensor (LFB) assay 26 (MCDA-LFB) was established to identify the mcr-1 gene with simpleness, rapidity, 27 specificity and sensitivity. The MCDA-LFB assay was performed at a isothermal temperature 28 (63°C) for only 30 min during the amplification stage, and the reaction products were directly 29 identified by using LFB which obtained the result with 2 min. The entire process of 30 experiments, from templates extraction to result judging, was accomplished less than 60 min. 31 For the analytical specificity of this method, all of the 16 mcr-1-producing strains were 32 positive, and all of the non-mcr-1 isolates got the negative results. The sensitivity of 33 mcr-1-MCDA-LFB assay was as little as 600 fg of plasmid DNA per reaction in pure culture, 34 and approximately 4.5×10 3 CFU/mL (~4.5 CFU/reaction) in fecal samples spiked with 100 35 μl of strains. Therefore, this technique established in the present study is suitable for the 36 surveillance of mcr-1 gene in clinic and livestock industry. 37 38 3
A loop-mediated isothermal amplification assay combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) was established for the rapid and accurate detection of the mobilized colistin resistance gene (mcr-1), which causes the loss of colistin antibacterial efficacy in clinical treatments. The amplification stage of the assay was completed in 60 min at 63°C, and the reaction products could be visually detected by employing the LFB, which provided a fast (within 2 min) and objective method to evaluate the amplification results. The LAMP assay amplified the target sequences of mcr-1 with high specificity. In pure strains, the detection limit of the LAMP-LFB assay was 360 fg plasmid DNA/reaction, and in spiked feces samples the value was approximately 6.3×103 CFU/mL (~6.3 CFU/reaction), which was tenfold more sensitive than the PCR assay. The results show that the developed LAMP-LFB assay will be a worthy tool for the simple, rapid, specific, and sensitive detection of mcr-1 gene in clinical settings and resource-limited areas.
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