Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

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

Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance

“…All Flavobacterium psychrophilum stains used in this study are described in Table 1 (Warsen et al 2004) and 8 of the strains were also confirmed by a nested PCR assay (Baliarda et al 2002) as part of a separate study (K. Cain & D. Call unpubl. data).…”

“…A microarray composed of rDNA oligonucleotides from 15 fish pathogens (Warsen et al 2004) was modified by the addition of a new oligonucleotide probe specific to a 16S rRNA gene polymorphism identified in Flavobacterium psychrophilum strain CSF 259-93 ( Fig. 1; GAAACACTCG-GTCGTGACCGAGCT).…”

“…This latter study also shows that the majority of polymorphisms are concentrated in the variable stemloop region of bacterial 16S rDNA. One of these regions, Variable Region 3 (Baker et al 2003), is the area targeted by our fish pathogen microarray (Warsen et al 2004). The high degree of variation in these variable regions is one reason they are attractive for identifying species-specific markers for pathogen detection.…”

Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array

“…This method enabled the identification of Vibrio and Photobacterium at the species level within a day including the PCR amplification step. Warsen et al (2004) developed a DNA array based on 16S ribosomal RNA gene for the discrimination of fish pathogens. By this method, 15 fish pathogens were simultaneously detectable, but only V. salmonicida was included in the experiment.…”

“…Because of their usefulness, DNA arrays have been utilized for bacterial identification (Call, 2005). Warsen et al (2004) developed a DNA array based on 16S ribosomal RNA gene for the discrimination of fish pathogens. Gonzalez et al (2004) reported the detection of marine fish pathogenic bacteria using multiplex PCR and DNA microarray.…”