RESUMEN
BACKGROUND: Recognized as a resistance mechanism responsible for the emergence and prevalence of antimicrobial resistance, integron is 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" In this study, detection assays based on loop-mediated isothermal amplification (LAMP) methodologies targeting on class 1 to class 3 integrase genes was developed and evaluated. METHODS: LAMP methodology was employed to develop novel detection assays on class 1, 2 and 3 integrons. Firstly, this protocol was specifically designed to detect such integrons by targeting integrase genes intI1, intI2 and intI3. Development, evaluation and optimization of such LAMP assays was studied, including the reaction temperature, volumn, time, sensitivity and specificity of both primers and targets. A total of 1082 strains, including 397 integron positive and 685 integron negative microorganisms, were included for the application verification of the established LAMP assays. RESULTS: The indispensability of each primer was confirmed, and the optimal amplification was obtained under 63 °C for 45 min, with 25 µl reaction found to be the most cost-efficient volume. As application was concerned, all of the 397 integron-positive isolates yielded positive amplicons and other 685 integron-negative bacteria were negative for the integron-LAMP assays, revealing totaling 100% detection rate and specificity. CONCLUSIONS: The established integron-LAMP assays was demonstrated to be a valid and rapid detection method for integrons screening, which may aid in both the laboratory and clinical integron screening for microorganisms.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Integrones/genética , Secuencias Repetitivas Esparcidas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Antibacterianos , Bacterias/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Calor , Integrasas/clasificación , Integrasas/genética , Sensibilidad y Especificidad , Virulencia/genéticaRESUMEN
In this study, a number of frequently detected gene cassettes from bacterial integrons have been detected and characterized by rapid and simple loop-mediated isothermal amplification (LAMP) assays. Six gene cassettes commonly found in class 1 integrons were studied, including dfrA12, dfrA17, aadA2, aadA5, orfF, and blaVIM2. Primers design, sensitivity, specificity, optimization of each LAMP assay, as well as application of the developed 6 individual LAMP assays on a large scale of bacteria, had been conducted. The optimal amplification was obtained with temperature as 65 °C, reaction time span as 45 min and volume as 25 µl. For application, 272 isolates with various gene cassettes yielded expectable positive amplicons and other 685 integron-negative bacteria showed negative results for the LAMP assays, totaling 100% detection rate and specificity.
Asunto(s)
Bacterias/genética , Genes Bacterianos/genética , Integrones/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Cartilla de ADN , ADN Bacteriano , Calor , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The 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. RESULTS: In 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%. CONCLUSIONS: The 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.