Agricultural and Biosystems Engineering Publications
Campus Units
Agricultural and Biosystems Engineering
Document Type
Article
Publication Version
Accepted Manuscript
Publication Date
9-1-2018
Journal or Book Title
FEMS Microbiology Ecology
Volume
94
Issue
9
Research Focus Area(s)
Biological and Process Engineering and Technology
DOI
10.1093/femsec/fiy130
Abstract
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.
Copyright Owner
FEMS
Copyright Date
2018
Language
en
File Format
application/pdf
Recommended Citation
Stedtfeld, Robert D.; Guo, Xueping; Stedtfeld, Tiffany M.; Sheng, Hongjie; Williams, Maggie R.; Hauschild, Kristin; Gunturu, Santosh; Tift, Leo; Wang, Fang; Howe, Adina; Chai, Benli; Cole, James R.; Tiedje, James M.; and Hashsham, Syed A., "Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements" (2018). Agricultural and Biosystems Engineering Publications. 951.
https://lib.dr.iastate.edu/abe_eng_pubs/951
Included in
Biological Engineering Commons, Bioresource and Agricultural Engineering Commons, Genetics and Genomics Commons, Microbiology Commons
Comments
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 https://doi.org/10.1093/femsec/fiy130 and doi: 10.1093/femsec/fiy130. Posted with permission.