荧光定量PCR法检测副溶血弧菌tlh和tdh基因的表达差异

Detection of tlh and tdh expression differences in Vibrio parahaemolyticus by real-time quantitative PCR

摘要:

副溶血弧菌是广泛存在于近海区域, 盐湖和海产品中的食源性致病菌, 会引起大规模的食物中毒。TLH (不耐热溶血毒素)和TDH (耐热直接溶血毒素)是副溶血弧菌最主要的毒力基因, 通过比较毒力基因的表达量可以间接比较同种菌株在不同应激条件下以及不同菌株之间的毒力差异。本文以在不同条件下培养的3株Vp为材料, 分别提取其总RNA, 以16S rRNA为内标基因, 运用荧光定量PCR技术检测副溶血弧菌TLH和TDH基因在不同应激条件下的表达差异。结果表明: 不同菌株和同种菌株在不同应激条件下tlh、tdh基因表达差异均显著; tlh的最适表达条件分别为5%盐度和20 °C; tdh的最适表达条件分别为1%盐度和25 °C。运用SPSS软件对实验结果进行统计学分析表明: 菌株对tlh表达的影响大于盐度大于温度; 菌株对tdh表达的影响大于温度大于盐度。

Abstract:

Vibrio parahaemolyticus is a foodborne pathogenic bacteria which could be widely found in coastal areas, salt lakes and seafoods, it could cause large-scale food poisoning. tlh (Themolabile hemolysin) and tdh (Thermostable direct hemolyticus) are two of the most important virulence genes of Vp, we can compare the virulence of strains which in different stress conditions, as well as differences strains by comparing the expression levels of tlh and tdh indirectly. In this paper, three kinds of Vp cultured under different conditions, total RNA were extracted from them, 16S rRNA as an internal standard gene, using real-time quantitative PCR (RT-PCR) detect Vp’s tlh and tdh gene expression under different conditions. The results showed that gene tlh and tdh expression difference were significant in different strains and in same strains that under different condition, tlh optimal expression conditions were 5% salinity and 20 °C; tdh optimum expression conditions were 1% salinity and 25 °C. The use of SPSS software for statistical analysis of experimental results show that: factor that affect the expression of tlh is strain greater than salinity greater than temperature; factor that affect the expression of tdh is strain greater than temperature greater than salinity.