Multiple Cross Displacement Amplification Coupled With Gold Nanoparticles-Based Lateral Flow Biosensor for Detection of the Mobilized Colistin Resistance Gene mcr-1.

Lin Gong,Jie Che,Huiqiong Xu,Xiaoli Liu,Juan Li,Jiansheng Liang,Ernan Liu

doi:10.3389/fcimb.2019.00226

Abstract

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 × 103 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.

Highlights

The rapid increase of carbapenem-resistant Enterobacteriaceae (CRE) expressing Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-blactamase (NDM) and oxacillinase (OXA) OXA-48 has risen serious concerns in clinic
Positive reaction appeared with DNA from mcr-1producing E. fergusonii (ICDC-ZG2016M34-3), but not with NDM-1-positive E. coli (WHCDC-WH67), KPC-2-producing K. pneumoniae (WHCDC-WH108), and the blank control (Figure 2)
To optimize the reaction temperature of multiple cross displacement amplification (MCDA)-lateral flow biosensor (LFB) assay during the amplification stage, the plasmid DNA of E. fergusonii (ICDC-ZG2016M34-3) at the level of 6 pg per reaction was used as the templates

Summary

Introduction

The rapid increase of carbapenem-resistant Enterobacteriaceae (CRE) expressing Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-blactamase (NDM) and oxacillinase (OXA) OXA-48 has risen serious concerns in clinic. Since a new mobilized colistin resistance gene, mcr-1, carried by plasmid in an Escherichia coli was first reported in China in 2015 (Liu et al, 2016), which has been identified in numerous countries. The mcr-1-positive bacterial species include Salmonella enterica, E. coli, Escherichia fergusonii, Enterobacter aerogenes, K. pneumoniae, Citrobacter braaki, and Klebsiella aerogenes (Doumith et al, 2016; Li et al, 2016; Stoesser et al, 2016; Zeng et al, 2016; Sennati et al, 2017; Wang et al, 2017a, 2018a). The horizontal transfer of mcr-1 gene causing inflation of colistin-resistant isolates will lead to the shortage of effective measures for treating infections with multidrug-resistant bacteria. A rapid, sensitive and specific diagnostic assay for mcr-1 detection is imperative to devise

Methods

Results

Conclusion