P3-58 Multiplex PCR for Detection of Vibrio spp. and 5 Pathogenic Vibrio Species Using Primers Designed by Comparative Genomics

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Shin-Young Lee, Kyung Hee University, Yongin, South Korea
Jio Ryu, Kyung Hee University, Yongin, South Korea
Hyun Joong Kim, Iowa State University, Ames, IA
Hae-Yeong Kim, Kyung Hee University, Yongin, South Korea
Introduction: Vibrio spp. is the major foodborne pathogen and 11 Vibrio species are known as pathogenic species causing Vibrio infections represented by V. cholera, V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. mimicus. While genetic markers were limited as virulence factor genes in previous PCR for Vibrio diagnostics, comparative genomics could suggest primer sets developing a multiplex PCR with increased range of Vibrio species and enhanced specificity.

Purpose: The purpose of our study is to develop a Vibrio multiplex PCR and to evaluate its specificity with various Vibrio strains suggesting novel diagnostics of Vibrio spp. and five major Vibrio species.

Methods: Total 30 genome sequences of Vibrio were subjected through comparative genomics to screen specific genes for Vibrio spp. and five major pathogenic Vibrio species. Primer sets were designed and each primer sets were reacted with various DNAs of Vibrio type strains and other non-Vibrio pathogenic bacteria. A multiplex PCR was developed and evaluated with various Vibrio strains isolated from Korean local areas.

Results: Primers designed from Vibrio genome sequences showed the specificity on their target Vibrio spp. and species by PCR with type strains of Vibrio and non-Vibrio. A multiplex PCR was developed by 6 primer sets enabling detection of Vibrio spp. and five pathogenic Vibrio species and its specificity was confirmed with various Vibrio/non-Vibrio type strains. Total 117 Vibrio isolates were identified as Vibrio spp., V. parahaemolyticus, V. vulnificus and V. alginolyticus. And the specificity of the multiplex PCR was confirmed by sequencing the PCR products.

Significance: The developed multiplex PCR was proved its discriminating ability with various Vibrio type strains and isolates. We expect that this multiplex PCR could be applied to the accurate monitoring Vibrio species with advantages to protect human health against Vibrio infection.