Identification And Evaluation Of New Molecular Targets For The Detection Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a well-recognized human pathogen, which is widely distributed in marine and estuarine environments. This organism can cause gastroenteritis through ingestion of raw or partially cooked seafood. More attention has been paid to the increasing reports of disease outbreaks in humans, and the correct identification and rapid detection of this organism is of great importance.The identification of V. parahaemolyticus by traditional microbiological detection methods is time consuming and usually with a low sensitivity. Polymerase chain reaction (PCR) method, which is obviously rapid and highly specific, is extensively used to detect a number of pathogens. The target genes and their primers play an important role in PCR assay, which affect the specificity and sensitivity of this method. For V. parahaemolyticus, it was currently reported that the target genes in PCR methods were basically gyrB gene, tl, tdh and trh genes and toxR gene. All of them have been used for many years, but they are not represented very specific in PCR experiments. Fortunately, hundreds of bacterial genomes including one V. parahaemolyticus strain and other 11 Vibrionaceaes strains have been sequenced and published in the past decades, which would be a great pool of information resources to mine a relatively specific and new genes by comparison of these sequences.In this study, a genome comparison method was used to identify specific PCR target sequences of Vibrio parahaemolyticus, and its CDS value was compared with other 139 bacterial genomes. It was found that 20 genes of V. parahaemolyticus were relatively specific according to their E-value in BLAST, and 4 of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from 9 V. parahaemolyticus strains, whereas there were no amplification products from 9 other Vibrionaceae strains and 4 non-Vibrionaceae strains. The detection limit of PCR method was 13.6 fg/μL for DNA and 2.17×104 cfu/mL for culture of V. parahaemolyticus.