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NCUEIR > College of Science > Department of Biology > Periodical Articles > Item 987654321/12231

Please use this identifier to cite or link to this item: http://ir.ncue.edu.tw/ir/handle/987654321/12231

Title:	Detection of Pathogenic Bacteria in Shellfish Using Multiplex PCR Followed by CovaLinkTM NH Microwell Plate Sandwich Hybridization
Authors:	Lee, Chi-Ying;Panicker, Gitika;Bej, Asim K.
Contributors:	生物學系
Keywords:	Sandwich hybridization;Multiplex PCR;Shellfish;Pathogen;CovaLinkk NH
Date:	2003-05
Issue Date:	2012-07-03T04:02:47Z
Publisher:	Elsevier
Abstract:	Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA–DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLinkk NH microwell plate sandwich hybridization using phophorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 102 cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.
Relation:	Journal of Microbiological Methods, 53(2): 199-209
Appears in Collections:	[Department of Biology] Periodical Articles

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