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P1-61 Single Laboratory Validation of a Vibrio TaqMan qPCR Assay for Identification of Vibrio Isolates

Monday, August 4, 2014
Exhibit Hall D (Indiana Convention Center)
Paul Browning, New Mexico State University, Las Cruces, NM
Lyssa White, New Mexico State University, Las Cruces, NM
Juan Olea, New Mexico State University, Las Cruces, NM
Ruiqing Pamboukian, U.S. Food and Drug Administration, Rockville, MD
Ruben Zapata, New Mexico State University, Las Cruces, NM
Willis Fedio, New Mexico State University, Las Cruces, NM
Introduction: Vibrio cholerae (Vc), V. parahaemolyticus (Vp), and V. vulnificus (Vv) are well-documented human pathogens associated with seafood consumption.   For detection of vibrios, 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. This study compares the Life Technologies multiplex Vibrio qPCR assay with the BAM procedure for identification of Vibrio isolates.

Purpose: To evaluate real time PCR for rapid identification of vibrios.

Methods: Vibrio cultures were grown in APW overnight at 35°C, streaked onto selective agars and confirmed biochemically as Vc, Vv, or Vp using methods described in the BAM including API20E biochemical identification and conventional PCR for Vc, Vv, and Vp as outlined in the BAM.  For the Vibrio TaqMan Assay, Vibrio isolates were grown on T1N0 agar (Vc) or T1N3 agar (Vp and Vv).  To prepare template, a well isolated colony was suspended in saline, boiled for 10 min., centrifuged 15,000 x g for 3 min. and the supernatant was used for vibrio detection.  Template was diluted (1:100) for multiplex qPCR detection of Vc, Vv, and Vp as described by the manufacturer. 

Results: The Vibrio TaqMan assay correctly identified 50/52 Vc isolates while the BAM procedure (API 20E) identified all 52.  For Vv, 50/51 isolates were positive by the Vibrio qPCR assay, 45/51 by API20E and 50/51 by the FDA BAM PCR.  Both the Vibrio qPCR assay and the BAM PCR confirmed 50/50 isolates tested while the API20E identified 49/50 Vp isolates correctly.  Of the 45 near-neighbor Vibrio isolates, as identified by API20E, none were detected by the Vibrio qPCR assay.

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

Back to: Poster Session - Applied Laboratory Methods, Produce, Low-water Activity, Beverages and Water
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