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. 2012;12(3):2710-28.
doi: 10.3390/s120302710. Epub 2012 Feb 29.

Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray

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Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray

Chin-I Chang et al. Sensors (Basel). 2012.

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 10(3) 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.

Keywords: 16S rDNA; fish pathogen detection; naked-eye reading microarray.

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Figures

Figure 1.
Figure 1.
Hybridization and colorization diagram for fish pathogen probes. (A) Microarray map. Dots indicate the spotted position of each probe. 1: EV71 (positive control for hybridization); 2: Aehy; 3: Edta; 4: poly(A) (negative control); 5, 6 & 7: blank, with no spotted probes; 8: Flco; 9: Laga; 10: Vian; 11: Phda; 12: blank; 13: U735 (positive control for PCR); 14: Psan; 15: Stin; 16: U1352 (positive control for PCR). (B) Detection and typing results on the microarray. a: Positive and negative controls on corners; b: A. hydrophila; c: E. tarda; d: F. columnare; e: L. garvieae; f: V. anguillarum; g: P. damselae; h: P. anguilliseptica; i: S. iniae.
Figure 2.
Figure 2.
Multiplex hybridization of the 12 probes with the DNA amplicon from the sample with mixtures of fish kidney and the eight target pathogens. The spotted position of each probe was the same with Figure 1.
Figure 3.
Figure 3.
Detection limit assay on the microarray with serially diluted cultures of Edwardsiella tarda. Row 1: dotting with the probe U735. Row 2: dotting with the probe Edta. Row 3: dotting with the probe poly(A). Row 4: dotting with the probe U1352. E. tarda suspensions in serial dilutions were used as samples for microarray detection. Bacterial concentrations (CFU/mL): (a) 4 × 109; (b) 4 × 108; (c) 4 × 107; (d) 4 × 106; (e) 4 × 105; (f) 4 × 104; (g) 4 × 103; (h) 4 × 102; (i) 4 × 101. Positive signals resulted from four independent PCR amplicons.

References

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