Development of a multiplex polymerase chain reaction (m-PCR) for the detection and identification of virulent and avirulent forms of Vibrio parahaemolyticus

Abstract

This study developed a new multiplex polymerase chain reaction (m-PCR) for rapidly detecting clinically essential strains of V. parahaemolyticus. This enables the detection of total and potentially virulent strains. The m-PCR was developed by targeting the species-specific transcriptional regulator toxR gene, and sequences for an outer membrane protein and a hypothetical protein encoded by omp and htp, respectively. The omp and htp sequences were discovered originally by randomly amplified polymorphic DNA (RAPD)-PCR. The m-PCR was performed on V. parahaemolyticus isolates, 23 clinical and 32 environmental. The toxR gene 367 bp fragment amplification was found in all V. parahaemolyticus tested; 17 out of 23 clinical isolates (73.91%) showed amplification of the omp and htp. Four isolates showed amplification of the omp gene sequence but not the htp gene and 2 isolates exhibited amplification for htp but not for omp. Therefore, both sequences for omp and htp must be targeted by PCR to detect all potentially virulent strains. Of the other species tested, no amplification was seen. This study confirms that RAPD-PCR helps differentiate virulent and avirulent forms. This allowed the development of an m-PCR for identifying V. parahaemolyticus and detecting virulent forms. Keywords: Multiplex PCR, RAPD-PCR, Vibrio parahaemolyticus