Genotypic Analyses of Vibrio parahaemolyticus and Development of a Pandemic Group-Specific Multiplex PCR Assay

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Abstract
A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the “pandemic group”) were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS / new or orf8 , that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin ( tdh ) gene also belonged to this cluster and possessed the toxRS / new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS / new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS / new sequence (651 bp).