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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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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.
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f1-sensors-12-02710: 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.

Mentions: Spotting 10 μM of each probe to each specific position on the microarray organic polymer substrate (patent no. US-7109024, supplied with the DR. Chip DIY Kit™, DR. Chip Biotechnology, Inc., Miao-Li, Taiwan) was performed using a contact spotting machine (DR. Fast Spot™; DR. Chip Biotechnology, Inc.), and immobilization using a UV crosslinker (Spectroline XLE-1000; Spectronics Corp., New York, USA) with 0.8 J/cm2 for 10 min. A schematic diagram of the probe position on the microarray is illustrated in Figure 1(a). Hybridization and colorimetric development were performed using the DR. Chip DIY Kit™ (DR. Chip Biotechnology, Inc.), and all of the reagents including DR. Hyb™ Buffer, Strep-AP, wash buffer, NBT/BCIP and detection buffer were supplied with the Kit. In brief, 15 μL of PCR amplicons were mixed with 200 μL DR. Hyb™ Buffer (DR. Chip Biotechnology, Inc.; 6× SSC, 5× Denhardt’s reagent, 0.5% SDS, 100 μg/mL salmon sperm DNA), denatured in boiling water for 5 min, and immediately chilled on ice for 5 min. The hybridization mixture was transferred to the chip well, incubated at 55 °C with vibration for 60 min, and washed twice with wash buffer (DR. Chip Biotechnology, Inc.; 0.1 M maleic acid, 0.15 M NaCl, pH 7.5). The chip was then added to 0.2 μL Strep-AP (DR. Chip Biotechnology, Inc.; 0.5 μL/mL in blocking buffer) and 200 μL blocking reagent (Roche GmbH, cat. no. 11096176001; 1%). at room temperature (25–35 °C) for 30 min and washed twice again with wash buffer. The colorimetric reaction was implemented by adding 4 μL NBT/BCIP and 196 μL detection buffer (DR. Chip Biotechnology, Inc.; 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) to the chip well, developing in the dark at room temperature for 5 min, and washing twice with distilled water. Hybridization results were indicated on the microarray as blue spots that could be read directly by the naked eye.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376613&req=5

f1-sensors-12-02710: 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.
Mentions: Spotting 10 μM of each probe to each specific position on the microarray organic polymer substrate (patent no. US-7109024, supplied with the DR. Chip DIY Kit™, DR. Chip Biotechnology, Inc., Miao-Li, Taiwan) was performed using a contact spotting machine (DR. Fast Spot™; DR. Chip Biotechnology, Inc.), and immobilization using a UV crosslinker (Spectroline XLE-1000; Spectronics Corp., New York, USA) with 0.8 J/cm2 for 10 min. A schematic diagram of the probe position on the microarray is illustrated in Figure 1(a). Hybridization and colorimetric development were performed using the DR. Chip DIY Kit™ (DR. Chip Biotechnology, Inc.), and all of the reagents including DR. Hyb™ Buffer, Strep-AP, wash buffer, NBT/BCIP and detection buffer were supplied with the Kit. In brief, 15 μL of PCR amplicons were mixed with 200 μL DR. Hyb™ Buffer (DR. Chip Biotechnology, Inc.; 6× SSC, 5× Denhardt’s reagent, 0.5% SDS, 100 μg/mL salmon sperm DNA), denatured in boiling water for 5 min, and immediately chilled on ice for 5 min. The hybridization mixture was transferred to the chip well, incubated at 55 °C with vibration for 60 min, and washed twice with wash buffer (DR. Chip Biotechnology, Inc.; 0.1 M maleic acid, 0.15 M NaCl, pH 7.5). The chip was then added to 0.2 μL Strep-AP (DR. Chip Biotechnology, Inc.; 0.5 μL/mL in blocking buffer) and 200 μL blocking reagent (Roche GmbH, cat. no. 11096176001; 1%). at room temperature (25–35 °C) for 30 min and washed twice again with wash buffer. The colorimetric reaction was implemented by adding 4 μL NBT/BCIP and 196 μL detection buffer (DR. Chip Biotechnology, Inc.; 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) to the chip well, developing in the dark at room temperature for 5 min, and washing twice with distilled water. Hybridization results were indicated on the microarray as blue spots that could be read directly by the naked eye.

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