Optimization and validation of a consolidated Set of TaqMan qPCR assays for the surveillance of clinically relevant antibiotic resistance genes in environmental matrices

Abstract

The continued rise of antibiotic resistance and adoption of the One Health approach necessitates reliable methods for detection and quantification of antibiotic resistance genes (ARGs) in complex environmental matrices. Here we present a consolidated set of TaqMan quantitative PCR assays for quantification of clinically relevant and emerging ARGs in complex environmental matrices.

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  We systematically designed five new primer sets, six TaqMan probes and verified and adapted four previously published relevant primer/probe sets from literature and evaluated their specificity in silico against current database.
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  For external quantification, two sets of gBlock standard libraries were designed. We experimentally validated the specificity, sensitivity, and efficiency of the assays with positive strain control DNA, negative strain control DNA, general no target controls, extraction blank controls, negative controls, and environmental test samples (i.e., metagenomic DNA from complex environmental matrices) to comprehensively assess each assays’ performance.
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  Optimization included iterative testing of both primer and probe concentration, annealing temperature, and annealing time. Results demonstrated robust and reliable detection and quantification of ARGs in clinical isolates and wastewater effluents with high sensitivity, specificity, and efficiency.

This makes the assays suitable for surveillance in wastewater or various environmental matrices, in support of efforts to mitigate dissemination of antibiotic resistance.