Detection of Antibiotic Resistance Genes Using Real-Time qPCR

Antibiotic resistance genes present in the bacterial community of SPF mice were assessed by microfluidic real-time PCR. Eleven antibiotic resistance predesigned TaqMan real-time PCR gene expression assays (Thermo Fisher Scientific Waltham, MA) were selected to target genes conferring vancomycin resistance in Enterococcus, and Type C β-lactamase, extended-spectrum β-lactamases, and carbapenem resistance in Enterobacteriaceae (Table 5). A total of 84 fecal DNA samples from WT and Nod2-KO mice from days 0, 14, 21, and 71 were assayed (10 KO and 11 WT per time point). PCRs were performed in a 10-μL volume with 4.5 μL of TaqMan Fast Universal PCR Master Mix (Applied Biosystems), 0.5 μL TaqMan assay, 1 μL fecal DNA template, and 4 μL PCR water. Assays were run on a ViiA 7 real-time PCR system (Applied Biosystems, Waltham, MA) using the following cycling conditions: 95°C for 20 seconds, followed by 45 cycles of 95°C for 1 second, and 60°C for 20 seconds. All assays were performed in duplicate with a positive control of bacterial DNA and a negative control of water. Real-time PCR data were analyzed with ViiA 7 RUO software, using the ΔΔCT-method as previously published.77 A Fisher exact test was performed to determine nonrandom significant associations between the genotypes, days, and antibiotics.