摘要
本研究开发、优化并证明了微流控定量聚合酶链反应( MF - qPCR )方法，用于同时定量39个抗生素抗性基因( ARGs )、5个重金属抗性基因、3个编码3种不同类型整合子整合酶的基因和16S rRNA基因(用于测量细菌总生物量)。由于MF - qPCR (几纳升)的模板体积比常规qPCR小得多，因此需要预扩增步骤来提高MF - qPCR方法的灵敏度和定量极限，使其与常规qPCR方法类似。MF - qPCR方法已成功应用于未处理的城市污水、处理后的城市污水和饮用水药品。与饮用水样品相比，经处理的城市污水样品所有基因的浓度都更高。类似地，与处理后的城市废水样品中的基因相比，未处理的城市废水样品对于除一个目标基因之外的所有目标基因具有更高的浓度。本研究建立的MF - qPCR方法为环境样品中多种ARG和其他基因提供了高精度的定量信息。
This study developed, optimized, and demonstrated a microfluidic quantitative polymerase chain reaction (MF-qPCR) method for the simultaneous quantification of 39 antibiotic resistance genes (ARGs), five heavy metal resistance genes, three genes encoding the integrase of three different classes of integrons, and 16S rRNA genes (used as a measure of total bacterial biomass). Because the volume of the template is much smaller with MF-qPCR (a few nanoliters) than with conventional qPCR, a preamplification step was needed to improve the sensitivity and the limits of quantification of the MF-qPCR method to be similar to those of conventional qPCR. The MF-qPCR method was successfully demonstrated on untreated municipal wastewater, treated municipal wastewater, and drinking water samples. The treated municipal wastewater samples had higher concentrations of all genes compared to those in the drinking water samples. Similarly, the untreated municipal wastewater samples had higher concentrations for all but one of the targeted genes compared to those in the treated municipal wastewater samples. The MF-qPCR method established in this study provides highly accurate quantitative information about numerous ARGs and other genes from environmental samples.
https://pubs.acs.org/doi/abs/10.1021/acs.estlett.7b00552
