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

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 × 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.

Visual multiple cross displacement amplification for the rapid identification of S. agalactiae immediately from vaginal and rectal swabs

“…MCDA products were usually analyzed by turbidity meter, intercalating dyes, agarose gel electrophoresis and LFB (Wang et al 2018 ; Gong et al 2019 ; Li et al 2020 ). Turbidity meter is an expensive device, which is not easy to obtain in the resource-limited hospitals, and the result is easily suffering from background interference.…”

<p>A Novel Detection of <em>Enterococcus</em> <em>faecalis</em> Using Multiple Cross Displacement Amplification Linked with Gold Nanoparticle Lateral Flow Biosensor</p>

“…For example, the dry block heater (Labnet, USA) is one of the suitable instruments, which is a portable, battery-powered equipment supporting 96-channel MCDA reactions per detection. Moreover, the universal isothermal amplification kits such as NEB Warmstart and Eiken Loopamp kits can be commercially obtained and applied for MCDA amplification, and the cost of MCDA reaction is estimated to be $3.5 USD, the LFB detection merely costs approximately $2 USD, which is cheaper than the common PCR-based methods 15,16. Additionally, this method can decrease labor costs because performing the MCDA-LFB assay does not need skilled technical personnel.…”

“…MCDA method can amplify target gene sensitively and efficiently, using a water bath or sample heater at a constant temperature 13,14. In the MCDA amplification system, six primers for amplification (C1, D1, C2, D2, R1, and R2), two displacement primers (F1 and F2), and two cross primers (CP1 and CP2), designed according to target DNA sequence, were used for the amplification 15,16. MCDA method is able to yield amplicons from as little as three microbes.…”

Current and emerging polymyxin resistance diagnostics: a systematic review of established and novel detection methods

“…A multiple cross displacement amplification (MCDA) method, coupled with gold nanoparticles-based lateral flow biosensor (LFB) assay for detecting mcr -1 gene was developed 81 . The MCDA reaction was performed on extracted DNA from 59 bacterial isolates, where each 25 µL reaction consisted of 12.5 µL reaction buffer, 1µL Bst DNA polymerase 2.0, 1µL colorimetric indicator, 1.6 µM of each cross primers, 0.4 µM of each displacement primers, 0.4 µM amplification primers and 1 µL DNA template 81 . The MCDA reaction systems were then subjected to isothermal temperature (63□) for 40min, after which the amplification products were analysed using 2% agarose gel electrophoresis, colorimetric indicator and LFB 81 .…”

“…The MCDA reaction was performed on extracted DNA from 59 bacterial isolates, where each 25 µL reaction consisted of 12.5 µL reaction buffer, 1µL Bst DNA polymerase 2.0, 1µL colorimetric indicator, 1.6 µM of each cross primers, 0.4 µM of each displacement primers, 0.4 µM amplification primers and 1 µL DNA template 81 . The MCDA reaction systems were then subjected to isothermal temperature (63□) for 40min, after which the amplification products were analysed using 2% agarose gel electrophoresis, colorimetric indicator and LFB 81 . For mcr -1 detection by LFB, 0.2 µL of the amplicons was added to the well of the sample pad, followed by the addition of three drops of running buffer (1% Tween 20 and 0.01 mol/L phosphate-buffered saline) 81 .…”