Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

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Abstract
We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenicVibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters.V. parahaemolyticushas been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) andtdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype ofV. parahameolyticusO3:K6. TotalV. parahaemolyticuswas targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFUV. parahaemolyticusper g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive fortlhand 4/33 samples that were positive fortdh. This assay specifically and sensitively detected total and pathogenicV. parahaemolyticusand is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

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