基于弧菌毒力基因多重PCR法的建立及其在副溶血弧菌毒力基因检测中的应用

Novel Multiplex Polymerase Chain Reaction Assay to Detect Virulence-related Genes in Vibrio parahaemolyticus

摘要:

针对副溶血弧菌常见的11种毒力基因（toxR、Collagenase、toxS、trh、tdh、tlh、UreR、FlaA、ompW、AspA、fur），建立了两套六重PCR检测体系，应用于副溶血弧菌环境分离株和水产品分离株的毒力基因分布情况调查。在调查的248株副溶血弧菌中，鞭毛丝蛋白基因FlaA、外膜蛋白基因ompW和铁吸收调节蛋白基因fur的分布最广（100%），其次为碱性丝氨酸蛋白酶基因AspA（99.60%），胶原蛋白酶基因Collagenase、不耐热性溶血毒素基因tlh以及毒力调控基因toxR和toxS的分布率均在90%以上且toxR和toxS的分布极为相似，尿素酶基因UreR的分布极少（1.21%），而耐热直接溶血素基因tdh和耐热相关溶血素基因trh在这248株副溶血弧菌中没有检出。本研究建立的多重PCR检测体系能快速、高效地检测多个毒力基因的分布情况，为副溶血弧菌的毒力机制研究和风险评估提供方法和依据。

Abstract:

Two multiplex polymerase chain reaction (PCR) assay systems were developed to detect 11 common virulence-related Vibrio parahaemolyticus (V. parahaemolyticus) genes (toxR, collagenase gene, toxS, trh, tdh, tlh, UreR, FlaA, ompW, AspA, and fur). The two systems were applied to investigate the distribution of these virulence genes in 248 strains of V. parahaemolyticus isolated from the environment and aquatic products. The results showed that FlaA, encoding flagellin protein; ompW, encoding the outer membrane protein; and fur, encoding the ferric uptake regulator were the most widely distributed (100%) genes, followed by the alkaline serine protease gene, AspA (99.60%). Genes with > 90% distribution included collagenase gene; tlh, encoding the thermolabile hemolysin; and the virulence regulatory genes, toxR and toxS, and tlh and toxR possessed similar distribution. The urease encoding UreR was distributed at a very low level (1.21%), while the thermostable direct hemolysin gene, tdh and the thermostable related hemolysin gene, trh were not detected in any of the 248 V. parahaemolyticus strains. The multiplex PCR assays established in this study can be kconsidered as a rapid and efficient method to investigate the distribution of virulence genes, which can provide data for virulence research and ris assessment regarding V. parahaemolyticus.