Evaluation of Typing of Vibrio parahaemolyticus by Three PCR Methods Using Specific Primers

Vibrio parahaemolyticus is a halophilic bacterium frequently involved in human outbreaks of seafood-associated gastroenteritis. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming. In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC). Typing patterns and clustering analysis indicated that these methods apparently differentiated V. parahaemolyticus strains from reference strains of interspecific Escherichia coli, V. cholerae, and V. vulnificus and were also valuable in subspecies typing of this pathogen. Forty domestic strains of V. parahaemolyticus, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively. The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping. REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.