qPCR Analysis of Antibiotic Resistance Genes and Mobile Genetic Elements

High-throughput quantitative PCRs (HT-qPCR) were conducted using WaferGen SmartChip Realtime PCR system that contained a total of 370 validated primer sets as reported previously (Muurinen et al., 2017). A threshold cycle value (C_T) of 31 was used as the detection limit to differentiate between positive amplification and primer–dimers (Su et al., 2015). One negative control with no DNA template added was included in each HT-qPCR run to eliminate false-positive detections. Amplicons with multiple melting curves were removed from the analysis. Three technical replicates of each sample above the detection limit were regarded as positive detection. The 2^–ΔCT method where ΔC_T = (C_T detected ARGs - C_T 16S rRNA gene) was used to calculate the relative abundances of ARGs and MGEs normalized to the 16S rRNA gene according to a comparative C_T method (Gou et al., 2018).

Fifteen genes of special concern ARGs including tetracycline resistance genes [tetG, tetL, tetZ, tetW, tetM, tetO, and tetB(P)], sulfonamide resistance genes (sul1, sul2, and sul3), macrolide resistance genes (ermB, ermC, and ermF), and beta-lactamase genes (bla_CTX and bla_TEM) were quantified by real-time quantitative PCR (RT-qPCR) analysis with a C1000^TM Thermal Cycler equipped with the CFX96^TM Real-Time system (Bio-Rad, United States). In addition, the 16S rRNA gene abundance, which has been used previously to assess the overall bacterial abundance, was quantified by using primer set 519F/907R so that the ARG abundance could be standardized against the bacterial populations. The qPCR system and primers sets as reported previously (Peng et al., 2017).