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against current database.•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.•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
Rathinavelu et al. (Thu,) studied this question.