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Sensors 2012, 12(3), 2710-2728; doi:10.3390/s120302710
Article

Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

1,†,* , 2,†
, 1
, 3
, 4
, 1
 and 5,6,7
1 Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, 199 Hou-Ih Road, Keelung 20246, Taiwan 2 Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Pei-Ning Road, Keelung 20224, Taiwan 3 Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, 1 Hseuh-Fu Rd., Nei-Pu Hsiang, Pingtung 91201, Taiwan 4 Department of Marine Biotechnology, National Kaohsiung Marine University, 142 Hai-Chuan Road, Kaohsiung 81157, Taiwan 5 Institute of Information Science, Academia Sinica, No. 128 Academia Rd., Sec. 2, Taipei 115, Taiwan 6 Institute of Fisheries Science, College of Life Science, National Taiwan University, No. 1, Roosevelt Rd., Sec 4, Taipei 10617, Taiwan 7 Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes, No. 35, Keyan Rd., Zhunan 350, Taiwan These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 6 February 2012 / Revised: 20 February 2012 / Accepted: 27 February 2012 / Published: 29 February 2012
(This article belongs to the Special Issue Biochips)
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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.
Keywords: fish pathogen detection; 16S rDNA; naked-eye reading microarray fish pathogen detection; 16S rDNA; naked-eye reading microarray
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Chang, C.-I.; Hung, P.-H.; Wu, C.-C.; Cheng, T.C.; Tsai, J.-M.; Lin, K.-J.; Lin, C.-Y. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray. Sensors 2012, 12, 2710-2728.

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