摘要
目的建立一种快速检测副溶血弧菌的实时荧光定量PCR方法。方法根据副溶血弧菌tdh基因设计合成引物及TaqMan探针,利用阳性质粒和模拟标本建立副溶血弧菌实时荧光定量PCR检测方法。结果以副溶血弧菌DNA为模板克隆了tdh基因,得到阳性质粒。利用阳性质粒建立了定量PCR方法 ,得到标准曲线y=-4.9197x+58.72,决定系数(R2)为0.9864。此方法具有很好的特异性,在对15种肠道致病菌的检测中,只有副溶血弧菌基因组DNA的扩增结果为阳性。对粪便和食物模拟标本进行检测,敏感性均为102cfu/μl。此方法重复性较好,对4种浓度(105、104、103、102cfu/μl)的菌液各进行3次检测,结果均为阳性,且Ct值的变异系数<2%。结论建立了一种检测副溶血弧菌的荧光定量PCR方法 ,该法的敏感性和特异均较好,适用于快速、准确地检测食品和粪便中的沙门菌。
Objective To develop a rapid and accurate method for detection of Vibrio parahaemolyticus by real-time fluorescent PCR assay. Methods According to tdh gene of V. parahaemolyticus,specific primers and probe were designed and synthesized. The genomic DNA contained in the boiled lysates of V. parahaemolyticus was used as a template for development of the PCR assay. The purified PCR products were used to prepare the positive plasmid. By using positive plasmid and simulated specimens,a new real time fluorescent PCR was developed. Purified DNA from V. parahaemolyticus strains and other bacteria were tested using standard PCR conditions. To test sensitivity,the positive plasmid was quantified and then 10 fold serially diluted. The experiment was repeated 3 times,it was showed that the robustness testing was very good. Results The tdh gene was amplified from genomic DNA of V. parahaemolyticus and cloned into pMD-18T to generate positive plasmid pMD-tdh. This plasmid was used to develop the PCR method and standard curve y=-4.9197x＋58.72,R2=0.9864. The PCR showed no cross-reactivity to other bacteria,and only the fluorescent amplification curve of V. parahaemolyticus was observed as a typical ＂S＂ shape,and the results of other 14 genera were negative. The limit of quantitative detection of this method was 102cfu for artificially contaminated dejecta and food samples. This method showed a good reproducibility. The CV of the results for 3 repeats was less than 2%. Conclusion A new real time PCR has been developed for rapid and specific detection of V. parahaemolyticus,suggesting such method may be useful in fast detection of Salmonella pollution in food and dejecta.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期969-972,共4页
Medical Journal of Chinese People's Liberation Army
基金
科技部传染病重大专项课题(2009ZX10004-205
2009ZX10004-103
2008ZX10004-015)
