Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA–DNA hybridization on a microarray

a.
The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
b.
College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

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Corresponding author: E-mail address: wshyyl@sina.com (Shihua Wang)

摘要

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Abstract: In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA–DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes (tlh, tdh and fla) that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin–conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains (tlh⁺/tdh⁺/fla⁺) and four known avirulent strains (tlh⁺/tdh⁻/fla⁺) of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains (tlh⁻/tdh⁻/fla⁺) of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria (tlh⁻/tdh⁻/fla⁻) gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.
- Multiplex PCR /
- Hybridization /
- Gene microarray

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参考文献(44)

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