Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood |
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Authors: | Chen Wanyi Yu Shuijing Zhang Chunxiu Zhang Jilun Shi Chunlei Hu Yu Suo Biao Cao Huan Shi Xianming |
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Institution: | (1) Joint Sino-US Food Safety Research Center & Bor Luh Food Safety Center, School of Agriculture & Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China;(2) National Engineering Center for Biochip at Shanghai, 151 Libing Rd., Shanghai, 201203, China;(3) Shanghai Entry–Exit Inspection and Quarantine Bureau, 1208 Minsheng Rd., Shanghai, 200135, China;(4) Department of Food Science and Technology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai, 200240, China; |
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Abstract: | In this study, a single base extension-tag array on glass slides (SBE-TAGS) microarray was established to detect the seven
leading seafood-borne pathogens, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio anguillarum, and Vibrio harveyi. Three multiplex PCR assays were developed to specifically target the following species with individual gene markers, which
are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus, and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi, and V. cholerae, respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with
Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with
the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally
contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified
as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray
hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry. |
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