Use of synthesized double-stranded gene fragments as qPCR standards for the quantification of antibiotic resistance genes.

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.