Abstract
We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.
Highlights
It may be assumed that fish are continually bathed in an aqueous suspension of microorganisms.Many of the members of the normal microflora of water can be bacterial fish pathogen candidates.Aeromonas hydrophila, an etiological agent of fish diseases, is considered as both a primary and secondary pathogen resulting in hemorrhagic septicemia [1]
To assess the overall detection limit of the microarray with purified genomic DNA, a serial dilution (100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg) of genomic DNA extracted from the eight pathogen collection strains (ATCC7966, ATCC15947, NCIMB2248, MT2055, ATCC19264, ATCC33539, NCIMB2248, and ATCC29178; Table 1) was used as the template for 16S rDNA PCR followed by hybridization to the microarray
The 16S rDNA amplicon was obtained by PCR using 16S universal primers 16S-F and 16S-R for each genomic DNA of 168 strains
Summary
It may be assumed that fish are continually bathed in an aqueous suspension of microorganisms. Outbreaks of disease caused by Vibrio anguillarum represent one of the most commonly occurring examples of vibriosis [22,23] This pathogen usually produces hemorrhagic septicemia [24], is distributed worldwide, and affects a wide range of fish and shellfish [25,26,27]. The pathogenic gram-positive cocci Lactococcus garvieae and Streptococcus iniae usually cause hyperacute and hemorrhagic septicemia in both freshwater and marine aquaculture species such as catfish [28], tilapia [29], trout [30], and yellowtail [31]. These pathogens cause massive mortality and large economic losses in fish farming every year. We demonstrate a naked-eye reading microarray system targeting 16S rDNA to identify eight common fish pathogens, obviating the need for expensive fluorescence detection facilities
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