摘要

由于可能转移到人类病原体，环境中存在的抗微生物抗性基因（ARG）会对人类健康构成风险。监视是减轻环境传播的一个组成部分。移动遗传元件1类整合子-整合酶基因( int1 )的定量已经被提议作为测量多个ARGs的替代物。通过采用新兴的核酸方法，例如环介导等温扩增( LAMP )，可以进一步简化这种指示基因的测量。在本研究中，设计并测试了LAMP测定法来估计int1基因的相对丰度，包括设计通用细菌16S rRNA基因测定法。在用已知菌株验证敏感性和特异性之后，使用从河流和湖泊样品中提取的DNA测试测定。结果表明，int1基因LAMP分析与ARG相对丰度( qPCR测定)之间存在显著的皮尔逊相关( R2 = 0.8 )。为了证明LAMP试验的稳定性，当地社区学院的志愿者本科生也在相对“未经培训”的人员手中使用手持实时DNA分析设备Gene - z进行试验。总体而言，结果支持使用inti 1基因作为ARGs的指示物，LAMP试验为志愿者监测专业实验室外环境样品的人为污染提供了机会。

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 (R2 = 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 intI1 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.

https://www.sciencedirect.com/science/article/pii/S030147971730