Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink™ NH microwell plate sandwich hybridization

Chi Ying Lee, Gitika Panicker, Asim K. Bej

Research output: Contribution to journalArticle

57 Citations (Scopus)

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 CovaLink™ 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.

Original languageEnglish
Pages (from-to)199-209
Number of pages11
JournalJournal of Microbiological Methods
Volume53
Issue number2
DOIs
Publication statusPublished - 2003 May 1

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All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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