In:
FEMS Microbiology Ecology, Oxford University Press (OUP), Vol. 94, No. 9 ( 2018-09-01)
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.
Type of Medium:
Online Resource
ISSN:
1574-6941
DOI:
10.1093/femsec/fiy130
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
2018
detail.hit.zdb_id:
1501712-6