抗生素耐药基因（ARGs）对各种环境基质的污染已成为对人类健康日益严重的威胁。为了定量分析ARGs的存在，需要一种灵敏、可靠的qPCR检测方法，能够从不同类型的DNA提取液中检测出不同的基因。本研究选择了14个ARGs作为靶基因，包括blaTEM、blaOXA-1和blaCTX-M，编码β-内酰胺类药物的耐药基因；ermB，编码大环内酯类药物的耐药基因；tetA、tetG、tetM、tetQ、tetW和tetX，编码四环素类药物的耐药基因；sulⅠ和sulⅡ，编码磺胺类药物的耐药基因；drfA1和drfA12 d，编码甲氧苄啶的耐药基因；integron基因intI 1和intI 2。利用分子生物学方法对化学合成的双链基因片段进行修饰，并将其用作实时PCR标准，同时建立了内部qPCR检测方法。利用天然质粒中的ermB基因对qPCR法和化学合成的ermB法进行了比较。此外，使用环境水、土壤和粪便样本验证所建立的qPCR分析。重要的是，该研究证明了快速合成的寡核苷酸作为ARGs分析的qPCR标准的有用性，并提供了与传统扩增子标准相当的灵敏度和可靠性。

      Pollution of various environmental matrices by antibiotic resistance genes (ARGs) has become a growing threat to human health. For the quantitative analysis of the presence of ARGs, there is a need for sensitive and robust qPCR assays which can detect various genes from different types of DNA extracts. Fourteen ARGs were selected as target genes in this study including: blaTEM, blaOXA-1 and blaCTX-M coded for resistance to β-lactams; ermB for macrolides; tetA, tetG, tetM, tetQ, tetW and tetX for tetracyclines; sul I and sul II for sulfonamides; drfA1 and drfA12 d for trimethoprim; and integron gene intI 1 and intI 2. Chemically synthesized double-stranded gene fragments were modified using molecular biology methods and used as real-time PCR standards as well as to establish in-house qPCR assays. The ermB gene from a naturally occurring plasmid was used to compare the performance of qPCR assay with the chemically synthesized ermB. Additionally, environmental water, soil and faeces samples were used to validate the established qPCR assays. Importantly, the study proves the usefulness of rapidly synthesized oligonucleotides serving as qPCR standards for ARG analysis and provides comparable sensitivity and reliability to a traditional amplicon standard.

       https://www.sciencedirect.com/science/article/pii/S0167701219303744?via%3Dihub