副溶血性弧菌热稳定直接溶血素基因转录水平差异研究

A study on difference of transcription level of thermostable direct hemolysin in Vibrio parahaemolyticus

目的运用实时荧光定量PCR检测24株副溶血性弧菌热稳定直接溶血素(TDH)基因转录水平差异。方法普通PCR法检测分离自患者、海产品和环境中的副溶血性弧菌tdh基因,tdh阳性菌株提取细菌总RNA后逆转录合成cDNA,检测无DNA污染后定量至同一浓度,采用实时荧光定量PCR同时检测cDNA产物中tdh基因和内标基因16 S rRNA的Ct值,ΔCt等于tdh的Ct值减去16 S rRNA的Ct值,以ΔCt值反应tdh相对于内标基因16 S rRNA的转录水平。结果 24株副溶血性弧菌的tdh Ct值、内标基因16 S rRNA Ct值和各样本两者之差的ΔCt值结果范围分别为18.04～25.95、8.30～10.93和8.28～15.34,ΔCt最大值与最小值差为7.06,最高转录水平菌株为最低菌株的133倍(ΔΔCt=27.06)。结论副溶血性弧菌tdh基因转录水平存在较大差异,该差异性形成的分子机制以及与该菌的致病性的关系有待进一步研究。

Objective To measure the transcription level of thermostable direct hemolysin gene （TDH）in 24 strains of Vibrio parahaemolyticus.Methods Total RNA was extracted from strains of Vibrio parahaemolyticus which were isolated from patients,seafood and environment.The RNA was proved TDH positive with routine PCR method;then the real -time fluorescent quantitative PCR was carried out to obtain the cycle of threshold （Ct）of THD and internal standard of 16s rRNA.Transcription level of THD compared with 16s rRNA was designated as ΔCt which was calculated as Ct value of THD minus Ct value of 16s rRNA.Results Ct values of THD,16s rRNA and the difference between them of the 24 strains was 18.04 ~25.95,8.30 ~10.93 and 8.28 ~15.34 respectively.The difference between the maximum and the minimum of ΔCt was 7.06;the highest transcription level was 133 （ΔΔCt =2^7.06 ）times of the lowest one.Conclusion A great difference of transcription level of THD in Vibrio parahaemolyticus has been proved and further study is needed to clarify the possible molecular mechanisms and relationship between the transcription level of THD and pathogenic mechanism.