Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements

Stedtfeld, Robert
Howe, Adina
Guo, Xueping
Stedtfeld, Tiffany
Sheng, Hongjie
Williams, Maggie
Hauschild, Kristin
Gunturu, Santosh
Tift, Leo
Wang, Fang
Howe, Adina
Chai, Benli
Cole, James
Tiedje, James
Hashsham, Syed
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Howe, Adina
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Agricultural and Biosystems Engineering

The high-throughput antibiotic resistance gene (ARG) qPCR array, initially published in 2012, is increasingly used to quantify resistance and mobile determinants in environmental matrices. Continued utility of the array; however, necessitates improvements such as removing or redesigning questionable primer sets, updating targeted genes and coverage of available sequences. Towards this goal, a new primer design tool (EcoFunPrimer) was used to aid in identification of conserved regions of diverse genes. The total number of assays used for diverse genes was reduced from 91 old primer sets to 52 new primer sets, with only a 10% loss in sequence coverage. While the old and new array both contain 384 primer sets, a reduction in old primer sets permitted 147 additional ARGs and mobile genetic elements to be targeted. Results of validating the updated array with a mock community of strains resulted in over 98% of tested instances incurring true positive/negative calls. Common queries related to sensitivity, quantification and conventional data analysis (e.g. Ct cutoff value, and estimated genomic copies without standard curves) were also explored. A combined list of new and previously used primer sets is provided with a recommended set based on redesign of primer sets and results of validation.


This is a pre-copyedited, author-produced version of an article accepted for publication in FEMS Microbiology Ecology following peer review. The version of record Stedtfeld, Robert D., Xueping Guo, Tiffany M. Stedtfeld, Hongjie Sheng, Maggie R. Williams, Kristin Hauschild, Santosh Gunturu et al. "Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements." FEMS Microbiology Ecology 94, no. 9 (2018) is available online at and doi: 10.1093/femsec/fiy130. Posted with permission.