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|Detection of Pathogenic Bacteria in Shellfish Using Multiplex PCR Followed by CovaLinkTM NH Microwell Plate Sandwich Hybridization
|Lee, Chi-Ying;Panicker, Gitika;Bej, Asim K.
|Sandwich hybridization;Multiplex PCR;Shellfish;Pathogen;CovaLinkk NH
|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.
|Journal of Microbiological Methods, 53(2): 199-209
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