The Development Of Probe Melting Curve Analysis For Molecular Identification Of 57 K-serogroups Of Vibrio Parahaemolyticus

Objective: In order to develop a low-cost,high-throughput,rapid and simple serotype identification method for V.parahaemolyticus Kserogroups,we developed a multiplex ligation reaction based on probe melting curve analysis(MLMA)for simultaneous identification of V.parahaemolyticus57 K-serogroups in three tubes.Methods:The previous whole genome sequencing of 418 strains was conducted and 39 K-serogroups specific sequences were obtained through the further analysis,and 18 K-serogroups sequences were downloaded and analyzed from the public databases.We obtained 57 K-serogroups specific genes sequences for designing hybridization ligation primers.Then single and multiplex systems were established sequentially based on the MLMA,and the specificity,reproducibility,minimum detection limit,and consistency rate of the multiplex system were evaluated.Results:The developed MLMA assay of all the hybridization ligation primers for V.parahaemolyticus 57 K-serogroups were able to specifically identify the corresponding K-serogroups DNA templates,and there was no amplification inhibition of the melting curve peaks and no cross-reactivity between different K-serogroups.In addition,the MLMA assay displayed high reproducibility,with the difference of Tm values of the same K-serogroups melting curve peaks within the intra-and inter-assay of less than 1℃,and the standard deviation and coefficients of variation of no more than 1℃ and 1%,respectively.The minimum detection limit of K-serogroups target genes was0.1～1.0 ng/μL.The MLMA assay detection results of 595 strains showed that the consistency rate with the traditional serotyping test was 96.97% and the kappa value was 0.936.The five rare K-serogroups and 18 strains serotypes that could not be identified by the traditional serotyping method can be identified by MLMA assay.Conclusion : The novel molecular identification system for the identification of 57 K-serogroups based on the fluorescent probe melting curve method in this study provides a simple,rapid,stable and promising method for the identification of V.parahaemolyticus K-serogroups serotyping.The assay can be applied to the detection of V.parahaemolyticus infection and outbreak monitoring,providing a rapid detection means for clinical differential diagnosis and treatment of V.parahaemolyticus infection.