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, 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 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was