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P2-159 Single Laboratory Validation of a Vibrio Assay for Identification of Vibrio Isolates

Tuesday, July 24, 2012
Exhibit Hall (Rhode Island Convention Center)
Willis Fedio, New Mexico State University, Las Cruces, NM
Jessica Jones, U.S. Food and Drug Administration, Dauphin Island, AL
Ruben Zapata, New Mexico State University, Las Cruces, NM
Paul Browning, New Mexico State University, Las Cruces, NM
Cecelia Garcia, New Mexico State University, Las Cruces, NM
Ruiqing Pamboukian, U.S. Food and Drug Administration, Rockville, MD
Angelo DePaola, U.S. Food and Drug Administration, Dauphin Island, AL
Introduction: Vibrio cholerae (Vc), V.  parahaemolyticus (Vp), and V. vulnificus (Vv) are well-documented human pathogens associated with seafood consumption. The FDA BAM recommends enrichment in alkaline peptone water (APW) followed by colony isolation on selective/differential agar. Biochemical testing or conventional PCR procedures are recommended for identification of suspect Vibrio isolates.

Purpose: This study compares the BAX Vibrio assay to the BAM procedure for identification of Vibrio isolates.

Methods: Vibrio cultures were grown in APW overnight at 35 °C, streaked onto selective agars and confirmed biochemically as Vc, Vv, or Vp using API20E.  For  the BAM PCR confirmation, a crude lysate was prepared by boiling the overnight APW enrichment and 2 µl was used as template for detection of Vv (519 bp species-specific fragment of vvh) and Vp assay (450 bp species-specific fragment of tlh ).  PCR products were visualized by gel electrophoresis as described in the BAM. For the BAX Vibrio assay, the manufacture instructions were followed for multiplex qPCR detection of Vc, Vv, and Vp as described by the manufacturer.

Results: The BAX Vibrio assay correctly identified 50/52 Vc isolates while API20E identified all 52. For Vv, 51/51 isolates were positive by the BAX Vibrio assay, 45/51 by API20E and 50/51 by the FDA BAM PCR. Both the BAX Vibrio assay and API20E identified all 55 Vp isolates correctly and the BAM PCR confirmed 54/55. Of the 30 near-neighbor Vibrio isolates, as identified by API20E, none were detected by the BAX Vibrio assay.

Significance: The results presented here demonstrate the BAX Vibrio assay is a reliable and rapid alternative to the BAM methods for identification of Vc, Vv, and Vp isolates.

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