Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

Author:

Ward Linda N.12,Bej Asim K.1

Affiliation:

1. Department of Biology

2. Department of Justice Sciences, University of Alabama at Birmingham, Birmingham, Alabama

Abstract

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 pathogenic Vibrio parahaemolyticus , including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has 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 ) and tdh -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 of V. parahameolyticus O3:K6. Total V. parahaemolyticus was 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 10 4 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 10 9 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh . This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference62 articles.

1. American Public Health Association. 1970. Recommended procedures for the examination of seawater and shellfish 4th ed. American Public Health Association Washington D.C.

2. Atlas, R. M. 1993. Handbook of microbiological media, p. 529. CRC Press, Boca Raton, Fla.

3. Atlas, R. M., and A. K. Bej. 1994. Polymerase chain reaction, p. 418-435. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. ASM Press, Washington, D.C.

4. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Smith, J. G. Sideman, and K. Struhl (ed.). 1987. Current protocols in molecular biology, p. 2.10-2.11. John Wiley & Sons, Inc., New York, N.Y.

5. Bates, T. C., and J. D. Oliver. 2004. The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus. J. Microbiol.42:74-79.

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