A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

Strauss, Christian; Endimiani, Andrea; Perreten, Vincent (2015). A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria. Journal of microbiological methods, 108, pp. 25-30. Elsevier 10.1016/j.mimet.2014.11.006

[img] Text
Strauss et al. 2015_Journal of Microbiological Methods 108_25–30.pdf - Published Version
Restricted to registered users only
Available under License Publisher holds Copyright.

Download (700kB) | Request a copy

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Veterinary Bacteriology > Molecular Bacterial Epidemiology and Infectiology
05 Veterinary Medicine > Research Foci > Host-Pathogen Interaction
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP)
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Veterinary Bacteriology

UniBE Contributor:

Strauss, Christian; Endimiani, Andrea and Perreten, Vincent

Subjects:

600 Technology > 630 Agriculture
500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0167-7012

Publisher:

Elsevier

Language:

English

Submitter:

Vincent Perreten

Date Deposited:

03 Feb 2015 17:12

Last Modified:

15 Oct 2017 02:44

Publisher DOI:

10.1016/j.mimet.2014.11.006

PubMed ID:

25451460

Uncontrolled Keywords:

DNA fragmentation, Detection, Drug resistance, Hybridization, Microarray, phi29 polymerase

BORIS DOI:

10.7892/boris.62542

URI:

https://boris.unibe.ch/id/eprint/62542

Actions (login required)

Edit item Edit item
Provide Feedback