Identification Of Common Vibrio Parahaemolyticus Serotypes Using The Multiplex Ligation Reaction Based On The Probe Melting Curve Analysis

The serotype is known as a biological phenotype of many pathogens which provides unequivocal detail associated with the antigenic reaction.Besides,serotyping is essential for pathogens identification,infectious disease outbreak investigation and tracing the source.Recently,Vibrio parahaemolyticus has been reported to be the leading cause of foodborne disease in China based on the laboratory-based foodborne disease surveillance systems during 2011-2016.However,the current methods for serotyping V.parahaemolyticus are laborious,time-consuming and limited.Therefore,it is of great significance to develop a rapid,high efficient and high throughput method for serotyping V.parahaemolyticus.In this study,we firstly developed the serotyping identification of V.parahaemolyticus by using the multiplex ligation-dependent probe melting curve analysis(MLMA).The results of this study are listed as follows:A novel method based on MLMA was developed for serotyping V.parahaemolyticus O-serogroups according to specific genes,which was found to simultaneously distinguish 12 O-serogroups with no cross-reaction in a single reaction within 3.5 hours.The limit of detection ranged from 0.1 ng/?L to 1.0 ng/?L at the DNA level and the coefficient of variation(CV)values of the intra-assay and inter-assay are no more than 1% via analytical studies.Furthermore,the clinical evaluation using 426 isolates proved a complete concordance of the assay and conventional serological assay,which the agreement is 100% and Kappa value was 1.0.A novel method based on MLMA was established for serotyping common ten V.parahaemolyticus K-serotypes according to K-antigen specific genes sequences of 446 whole genome sequence,which was capable of simultaneously distinguishing common ten K-serotypes with no cross-reaction in a single reaction within 3.5hours.The limit of detection ranged from 0.1 ng/?L to 1.0 ng/?L at the DNA level and the coefficient of variation(CV)values of the intra-assay and inter-assay are no more than 1% via analytical studies.To comprehensively validate the assay,481 clinical and seafood isolates were tested with the comparison of conventional serological assay.The result illustrated the assay had a specificity and reproducibility of 100%,with a sensitivity and agreement of 98.9% and 99.0%,respectively.Moreover,the Kappa correlation is 0.79 with respect to all tested isolates,which showed high agreement with the traditional assay.Overall,two new methods were designed for serotyping 12 V.parahaemolyticus Oserogroups and 10 common K-serotypes,which demonstrated the assay was rapid,high efficient and high throughput and was a useful tool for laboratory based surveillance of V.parahaemolyticus infection.