DETECTION OF MICROBIAL PATHOGENS IN SHELLFISH WITH MULTIPLEX PCR

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, Sal monella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemol yticus, respectively. Each of the five pairs of oligonucleotide primer s was found to support PCR amplifications of only its targeted gene. T he optimized multiplex, PCR reaction utilized a PCR reaction buffer co ntaining 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C . Oyster tissue homogenate seeded with these microbial pathogens was s ubjected to DNA purification by the Chelex(TM) 100 (BioRad) method. Th e sensitivity of detection for each of the microbial pathogens was les s than or equal to 10(1)-10(2) cells following a ''double'' multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb(TM) (BioRad) DNA-DN A hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The co lorimetric GeneComb(TM) assay avoids use of hazardous materials inhere nt in conventional gel electrophoresis and radioactive-based hybridiza tion methods. Multiplex PCR amplification, followed by colorimetric Ge neComb(TM) DNA-DNA hybridization, has been shown to be an effective, s ensitive, and rapid method to detect microbial pathogens in shellfish.