Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.
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.
Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, Keelung 20246, Taiwan. cichang@mail.tfrin.gov.tw
Chin-I
CI
Chang
Pei-Hsin
PH
Hung
Chia-Che
CC
Wu
Ta Chih
TC
Cheng
Jyh-Ming
JM
Tsai
King-Jung
KJ
Lin
Chung-Yen
CY
Lin
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D003124:Colorimetry; D017931:DNA Primers / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology; D005399:Fishes / Q000382:microbiology; D016680:Genome, Bacterial; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D019809:Streptavidin / Q000737:chemistry; D013778:Tetrazolium Salts / Q000737:chemistry
D017931:DNA Primers; D015342:DNA Probes; D012336:RNA, Ribosomal, 16S; D013778:Tetrazolium Salts; C042207:4-anisyltetrazolium blue; D019809:Streptavidin; D000469:Alkaline Phosphatase