Abstract
BackgroundThe occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.ResultsIn this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%.ConclusionsThe intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.
Highlights
The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently
Optimization of integron-screening loop-mediated isothermal amplification (LAMP) assays Based on the amplification principle, the specific LAMP reaction generated many ladder-like pattern bands on agarose gel due to its characteristic secondary structure, with sizes ranging from 193 bp for intI1, 148 bp for intI2 and 168 bp for intI3, respectively (Figure 2)
The LAMP product amplified at 65°C exhibited slightly larger amount of DNA amplicons when compared to other temperatures, which was consistent with previous studies [4,26,46]
Summary
The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied. A complete integron platform may comprise three basic genetic elements, the integrase gene (intI), recombination site attI and a promoter (Pc). Through specific excision and integration, gene cassettes become part of integron and mediate various function for the hosts, with resistance cassettes mostly identified [15,16,17]. Integrons are considered to be widely distributed and spread among clinical microorganisms and play a key role in the dissemination of such antimicrobial resistance, which may eventually contribute to the unleashing of “Super Bugs” [23,24,25]
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