Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers

Abstract

Antimicrobial resistance genes (ARGs) present in the environment pose a risk to human health due to potential for transfer to human pathogens. Surveillance is an integral part of mitigating environmental dissemination. Quantification of the mobile genetic element class 1 integron-integrase gene (intI1) has been proposed as a surrogate to measuring multiple ARGs. Measurement of such indicator genes can be further simplified by adopting emerging nucleic acids methods such as loop mediated isothermal amplification (LAMP). In this study, LAMP assays were designed and tested for estimating relative abundance of the intI1 gene, which included design of a universal bacteria 16S rRNA gene assay. Following validation of sensitivity and specificity with known bacterial strains, the assays were tested using DNA extracted from river and lake samples. Results showed a significant Pearson correlation (R-2 = 0.8) between the intI1 gene LAMP assay and ARG relative abundance (measured via qPCR). To demonstrate the ruggedness of the LAMP assays, experiments were also run in the hands of relatively "untrained" personnel by volunteer undergraduate students at a local community college using a hand-held real-time DNA analysis device - Gene-Z. Overall, results support use of the intli gene as an indicator of ARGs and the LAMP assays exhibit the opportunity for volunteers to monitor environmental samples for anthropogenic pollution outside of a specialized laboratory. (C) 2017 Elsevier Ltd. All rights reserved.