Journal article
Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays
Applied and environmental microbiology, Vol.70(7), pp.4216-4221
07/2004
Handle:
https://hdl.handle.net/2376/114086
PMCID: PMC444826
PMID: 15240304
Abstract
We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 10
6
genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.
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Details
- Title
- Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays
- Creators
- Adelaide E Warsen - Department of Veterinary Microbiology and PathologyMelissa J Krug - Department of Veterinary Microbiology and PathologyStacey LaFrentz - Department of Veterinary Microbiology and PathologyDanielle R Stanek - Department of Veterinary Microbiology and PathologyFrank J Loge - Department of Veterinary Microbiology and PathologyDouglas R Call - Department of Veterinary Microbiology and Pathology
- Publication Details
- Applied and environmental microbiology, Vol.70(7), pp.4216-4221
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- American Society for Microbiology
- Identifiers
- 99900548076901842
- Language
- English
- Resource Type
- Journal article