Detection of Microbial Pathogens in Shellfish with Multiplex PCR |
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Authors: | Cynthia W Brasher Angelo DePaola Daniel D Jones Asim K Bej |
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Institution: | (1) Department of Biology, The University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170, USA , US;(2) U.S. Food and Drug Administration, Gulf Coast Seafood Laboratory, Dauphin Island, AL 36528, USA , US |
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Abstract: | Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted
gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to
DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was
≤101–102 cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were
subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of
detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials
inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed
by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect
microbial pathogens in shellfish.
Received: 17 November 1997 / Accepted: 17 February 1998 |
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