海南地区哈维氏弧菌病原分子血清型特征及毒力相关基因鉴定研究

The most important bases of mariculture and hatchery in China are located in Hainan. In recent years, a novel pathogenic strain of Vibrio harveyi was the most serious bacterial pathogen of the mariculture system and hatchery factory in Hainan. However, its pathogenic mechanism are still unclear, and no measure can effectively control the epidemic caused by this pathogen. Therefore, it is necessary to identify the molecular epidemiological characteristics, virulence-related genes and the molecular biomarker for controlling the disease. In this study, RAPD, REP-PCR and ERIC-PCR will introduced to determine the molecular epidemiological characteristics of the V. harveyi strains isolated from the mariculture system and hatchery factory in Hainan. One of the typic wild virulent strain (WV) will be mutated by Ultraviolet and both of the mutant virulent strains (MV) and nonvirulent strains (MNV) will be selected by artificial tests. The genomes of WV, MV and MNV will be sequenced by Pacbio and Hiseq sequencing, respectively and the virulence-related genes of WV will be obtained thereafter. Finally, the molecular biomarker, the specific nucleic acid fragment among the virulence strains which presented the similar molecular epidemiological characteristics with WV, will be determined by Southern hybridization and a specific PCR detection method for the pathogen will be developed. The results of this study will be very helpful for revealing pathogenic mechanism, epidemiological investigation, pathogen tracking, diagnosis, discovering the transmission mechanism and formulating the prevention and control measures of the disease caused by V. harveyi in China.

海南是我国南方重要的海水动物苗种繁育和养成基地，哈维氏弧菌是该地区海水养殖和苗种繁育中最常见和危害最大的细菌性病原之一，且这些菌株可能是一类新的、致病机理不清的分子血清型毒力菌株。当前，对哈维氏弧菌病还缺少安全有效的防控方法。为此，本项目将采用RAPD、ERIC-PCR和REP-PCR技术相结合，确定申请人近年来从海南地区分离的哈维氏弧菌病原分子血清型特征；采用紫外突变技术诱发哈维氏弧菌病原典型菌株（WT）产生突变，比较WT、突变毒力株和突变非毒株的基因组差异，确定该地区典型菌株的毒力相关基因；采用Southern技术，确定该地区哈维氏弧菌病原的特异性分子标志并建立相应的PCR快速检测技术。本项目研究结果将为开展哈维氏弧菌分子致病机理研究，和海南乃至我国哈维氏弧菌病的流行病学调查、病原追踪、快速诊断以及探寻其传播机制和制订安全高效的哈维氏弧菌病控制措施奠定良好基础。

针对哈维氏弧菌为海南地区海水养殖和苗种繁育中最常见和危害最大的细菌性病原之一且分子血清型不清的问题，本课题采用RAPD和ERIC-PCR技术相结合，确定了项目组近年来从海南地区分离的哈维氏弧菌病原分子血清型特征；采用紫外突变技术诱发哈维氏弧菌病原典型菌株GDH11385产生突变，通过比较WT、突变毒力株（含毒力不变株和毒力增强株）和突变非毒株的基因组差异，基本确定了与运动、胞外结构、细胞膜形成、抵抗机制、次生代谢物合成和转运与催化活性相关的约40个基因为该地区典型菌株的毒力相关基因；采用基因序列分析技术，确定了该地区哈维氏弧菌主要的特异性分子标志，进一步建立相应的PCR快速检测技术。本项目研究结果为海南乃至我国哈维氏弧菌病的流行病学调查、病原追踪、快速诊断以及探寻其传播机制和制订安全高效的哈维氏弧菌病控制措施，和今后开展哈维氏弧菌分子致病机理研究奠定了良好基础。