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

Chang CI, Hung PH, Wu CC, Cheng TC, Tsai JM, Lin KJ, Lin CY - Sensors (Basel) (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, Keelung 20246, Taiwan. cichang@mail.tfrin.gov.tw

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.

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Related in: MedlinePlus

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.
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f3-sensors-12-02710: 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.

Mentions: For the purpose of directly detecting pathogens in suspension samples, serially diluted cultures of the eight target strains were tested using the microarray. All target pathogens were detectable at concentrations in the range 103–104 CFU/mL (Table 6). For example, E. tarda was selected as representative and serial dilutions prepared in the range 4 × 101–4 × 109 CFU/mL. The detection limit was as low as 4 × 103 CFU/mL (Figure 3).


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

Chang CI, Hung PH, Wu CC, Cheng TC, Tsai JM, Lin KJ, Lin CY - Sensors (Basel) (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376613&req=5

f3-sensors-12-02710: 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.
Mentions: For the purpose of directly detecting pathogens in suspension samples, serially diluted cultures of the eight target strains were tested using the microarray. All target pathogens were detectable at concentrations in the range 103–104 CFU/mL (Table 6). For example, E. tarda was selected as representative and serial dilutions prepared in the range 4 × 101–4 × 109 CFU/mL. The detection limit was as low as 4 × 103 CFU/mL (Figure 3).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, Keelung 20246, Taiwan. cichang@mail.tfrin.gov.tw

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.

Show MeSH
Related in: MedlinePlus