Design and validation of primer sets for the detection and quantification of antibiotic resistance genes in environmental samples by quantitative PCR

Abstract

Abstract The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Hence, it is vital to dispose of molecular tools that allow proper monitoring of antibiotic resistant genes (ARGs) encoding resistances to these important therapeutic compounds. 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 ARG’s biodiversity than those of several primers described to date. The adequate design and well performance of the new molecular tools were validated in vivo in six samples, retrieved from both natural and engineered environments. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), consistency across replicate experiments, and a wide dynamic range. The new methodology described here provide valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs in the environment by qPCR.