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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

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.
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f2-sensors-12-02710: 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.

Mentions: Results of the preliminary test for the applicability of the microarray is illustrated in Figure 2. The probes for all the 8 pathogens (A. hydrophila, E. tarda, F. columnare, L. garvieae, P. damselae, P. anguilliseptica, S. iniae and V. anguillarum) gave positive signals. This result demonstrated that the probes designed were specific to their corresponding species.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f2-sensors-12-02710: 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.
Mentions: Results of the preliminary test for the applicability of the microarray is illustrated in Figure 2. The probes for all the 8 pathogens (A. hydrophila, E. tarda, F. columnare, L. garvieae, P. damselae, P. anguilliseptica, S. iniae and V. anguillarum) gave positive signals. This result demonstrated that the probes designed were specific to their corresponding species.

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