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

Like Xu; Hannah Chen; Melisa Canales; Lena Ciric

doi:10.1016/j.mimet.2019.105670

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

J Microbiol Methods. 2019 Sep:164:105670. doi: 10.1016/j.mimet.2019.105670. Epub 2019 Jul 17.

Authors

Like Xu¹, Hannah Chen¹, Melisa Canales¹, Lena Ciric²

Affiliations

¹ Healthy Infrastructure Research Group, Department of Civil, Environmental & Geomatic Engineering, University College London, Gower Street, WC1E 6BT London, UK.
² Healthy Infrastructure Research Group, Department of Civil, Environmental & Geomatic Engineering, University College London, Gower Street, WC1E 6BT London, UK. Electronic address: l.ciric@ucl.ac.uk.

PMID: 31325465
DOI: 10.1016/j.mimet.2019.105670

Abstract

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: bla_TEM, bla_OXA-1 and bla_CTX-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.

Keywords: Antibiotic resistance genes; Chemically synthesized gene fragments; Gene cloning; qPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / pharmacology
Cloning, Molecular
DNA, Bacterial / isolation & purification
Drug Resistance, Microbial / genetics*
Environmental Monitoring
Feces / microbiology
Genes, Bacterial / genetics*
Molecular Biology / methods
Real-Time Polymerase Chain Reaction / methods
Real-Time Polymerase Chain Reaction / standards*
Reproducibility of Results
Sensitivity and Specificity
Soil
Soil Microbiology
Water Microbiology

Substances

Anti-Bacterial Agents
DNA, Bacterial
Soil