Antimicrobial resistance gene analysis

The ARG Sul1 and TetA were selected for qPCR (real-time PCR) analyses of sulfonamide (Sul) and tetracycline (Tet) resistance, respectively. These genes have been closely associated with class I integrons responsible for transfer of ARGs between bacteria (Cadena et al., 2018). DNA extractions were carried out using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. The qPCR of Sul1 and TetA were carried out using primers presented in Table 2. Primers were designed using AlleleID 7 (Premiere Biosoft, Palo Alto, CA) and were based on Sul1 and TetA nucleotide reference sequences {"type":"entrez-nucleotide","attrs":{"text":"NG_048098.1","term_id":"1035502269","term_text":"NG_048098.1"}}NG_048098.1 and {"type":"entrez-nucleotide","attrs":{"text":"NG_048153.1","term_id":"1035502315","term_text":"NG_048153.1"}}NG_048153.1, respectively, from the Bacterial Antimicrobial Resistance Reference Gene Database (NCBI). Reaction mixtures for qPCR included 10 µL of 2× QuantiTect SYBR Green PCR kit (Qiagen, Carlsbad, CA), 300 nM of each primer, and 1.5 µL of extracted DNA for a 20 µL total reaction volume. Thermocycling conditions were the same for Sul1 and TetA: 95 °C for 15 min; 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s; followed by a final melting from 65 to 95 °C, increased by 0.5 °C every 5 s. A standard curve was generated using serial dilutions (10² to 10⁷ copies) of gBlocks (Integrated DNA Technologies, Coralville, IA) designed from 250 and 500 bp fragments of the Sul1 and TetA genes, respectively (NCBI, 2019).

Sequences, target size, and melting temperature of primers used

¹T _m melting temperature.