@misc{10481/92706,
year = {2024},
month = {5},
url = {https://hdl.handle.net/10481/92706},
abstract = {The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human
health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to
receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence,
it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic
compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive
and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs
were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and
mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to
fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer
sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the
Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs’ biodiversity than
those of several primers described to date. The adequate design and performance of the new molecular tools were validated
in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of
the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980),
repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology
described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs
by qPCR in WWTPs and related environments.},
organization = {Funding for open access publishing: Universidad de Granada/CBUA.},
organization = {ECORESISTOME project
(A-RNM-62-UGR20) funded by Consejería de Universidad, Investigación
e Innovación de la Junta de Andalucia (Spain), and by “ERDF
A way of making Europe”},
organization = {Predoctoral
fellowship of the Carolina Foundation 2021},
organization = {Aid in the Hiring of Doctoral Research Personnel
Program of the Andalusian Government for postdoctoral contract},
publisher = {Springer Nature},
keywords = {Antibiotic resistance genes},
keywords = {Primer design},
keywords = {Assay optimization},
title = {Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR},
doi = {10.1007/s00248-024-02385-0},
author = {Pérez-Bou, Lizandra and González Martínez, Alejandro and Cabrera Contreras, Juan José and Juárez Jiménez, María José Belén and Rodelas González, María Belén and González López, Jesús Juan and Correa Galeote, David},
}