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

被引:23
作者
Chang, Chin-I [1 ]
Hung, Pei-Hsin [2 ]
Wu, Chia-Che [1 ]
Cheng, Ta Chih [3 ]
Tsai, Jyh-Ming [4 ]
Lin, King-Jung [1 ]
Lin, Chung-Yen [5 ,6 ,7 ]
机构
[1] Minist Agr, Aquaculture Div, Fisheries Res Inst, Keelung 20246, Taiwan
[2] Natl Taiwan Ocean Univ, Dept Aquaculture, Coll Life Sci, Keelung 20224, Taiwan
[3] Natl Pingtung Univ Sci & Technol, Dept Trop Agr & Int Cooperat, Pingtung 91201, Taiwan
[4] Natl Kaohsiung Marine Univ, Dept Marine Biotechnol, Kaohsiung 81157, Taiwan
[5] Acad Sinica, Inst Informat Sci, Taipei 115, Taiwan
[6] Natl Taiwan Univ, Inst Fisheries Sci, Coll Life Sci, Taipei 10617, Taiwan
[7] Natl Hlth Res Inst, Div Biostat & Bioinformat, Inst Populat Hlth Sci, Zhunan 350, Taiwan
关键词
fish pathogen detection; 16S rDNA; naked-eye reading microarray; 16S RIBOSOMAL-RNA; PSEUDOMONAS-ANGUILLISEPTICA STRAINS; PASTEURELLA-PISCICIDA INFECTION; REAL-TIME PCR; VIBRIO-ANGUILLARUM; MARINE FISH; SEQUENCE; DISEASE; HYBRIDIZATION; EELS;
D O I
10.3390/s120302710
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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.
引用
收藏
页码:2710 / 2728
页数:19
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