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
dc.contributor.department Agricultural and Biosystems Engineering 2018-07-31T18:53:25.000 2020-06-29T22:43:57Z 2020-06-29T22:43:57Z Mon Jan 01 00:00:00 UTC 2018 2019-09-01 2018-09-01
dc.description.abstract <p>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.</p>
dc.description.comments <p>This is a pre-copyedited, author-produced version of an article accepted for publication in <em>FEMS Microbiology Ecology</em> 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." <em>FEMS Microbiology Ecology </em>94, no. 9 (2018) is available online at and doi: <a href="" target="_blank">10.1093/femsec/fiy130</a>. Posted with permission.</p>
dc.format.mimetype application/pdf
dc.identifier archive/
dc.identifier.articleid 2234
dc.identifier.contextkey 12576048
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath abe_eng_pubs/951
dc.language.iso en
dc.source.bitstream archive/|||Sat Jan 15 02:34:19 UTC 2022
dc.source.uri 10.1093/femsec/fiy130
dc.subject.disciplines Biological Engineering
dc.subject.disciplines Bioresource and Agricultural Engineering
dc.subject.disciplines Genetics and Genomics
dc.subject.disciplines Microbiology
dc.subject.keywords high-throughput qPCR primer set
dc.subject.keywords antibiotic resistance gene
dc.subject.keywords mobile genetic element
dc.subject.keywords validation
dc.subject.keywords qPCR array
dc.subject.keywords SmartChip
dc.subject.keywords primer design
dc.subject.keywords EcoFunPrimer
dc.title Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements
dc.type article
dc.type.genre article
dspace.entity.type Publication
relation.isAuthorOfPublication e2017bbe-ba62-4969-946e-aaf072d8bb4f
relation.isOrgUnitOfPublication 8eb24241-0d92-4baf-ae75-08f716d30801
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