荧光定量PCR法检测副溶血弧菌tdh基因的表达差异

以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异,副溶血弧菌临床分离株的tdh mRNA平均表达量显著高于海产品分离株((57.2比13.8)。在pH4.0、0.5%和8%NaCl应激条件下,临床株ZJ2和海产品分离株FJ14A的tdh mRNA表达量显著高于对照组;另一海产品分离株KP34在8%NaCl条件下的表达量显著提高,而低pH应激时tdh mRNA的表达量显著降低。结果表明,不同副溶血弧菌分离株的tdh mRNA表达差异显著,临床分离株的tdh mRNA表达量总体上高于海产品分离株,副溶血弧菌在不同应激条件下主要表现为tdh mRNA表达上调。

Differential expression of tdh gene from the different Vibrio parahaemolyticus isolates as determined by real-time PCR

Real-time PCR method was used to detect the expression of the tdh gene in Vibrio parahaemolyticus isolates from different origins and under stress conditions using pvuA gene as the internal standard.The melting curve analysis of both pvuA and tdh genes indicated that the amplifications were specific.Expression of tdh gene from the clinical V.parahaemolyticus isolates were markedly higher than that from the environmental strains(57.2 vs 13.8),although considerable variations existed among different strains.The clinical isolate ZJ2 and seafood isolate FJ14A exhibited higher expression of tdh under stress conditions at pH 4.0,0.5% or 8% NaCl than the control at 3% NaCl and pH 7.4.The tdh mRNA expression of the seafood isolate KP34 was higher in 8% NaCl medium,but lower at pH 4.0 as compared to the control culture.The results indicated that the expression of tdh gene varied with different V.parahaemolyticus isolates.In general,clinical isolates had higher tdh expression than seafood islates,and stress conditions favored tdh expression as well.