12小时PCR技术快速检测沙门菌

Rapid detection of Salmonella by using a 12- hour PCR Method

[目的]本课题根据致痫性沙门菌的invA基因序列设计引物,进行PCR反应,扩增出389bp的特异性片断,从而检测出目标菌。[方法]将试验菌种BPV中非选择性增菌6h:45min沸水浴破细胞,释放染色体DNA制作模板:PCR扩增约3h（1:95℃5min预变性:2:95℃1.5min变性3:62℃lmin退火:4:72℃ 45S延伸:5:72℃7min延长其中4至2步循环35次）。[结果]所得扩增产物经1.5％的琼脂糖凝胶电泳约1h,紫外灯下可见扩增条带。[结论]通过调节Mg2+浓度,引物浓度和模板量和纯化方法,优化反应条件,初步建立了一种12h内快速检测沙门菌的方法,灵敏度可达40-50cfu/ml。

[Objective]A Twelve - Hour PCR- Based method for the detection of Salmonella spps. in food was established by applying the sequences of the chromosomal invA gene as primers.[Methods]Prepared samples from the 6 - h noneselective enrichment inoculation were amplified in 35 cycles at 95℃ for 1.5 min for denaturation, 1 min at 62℃ for annealing and 45s at 72℃ for extension. [Results]The products were analyzed by electrophoresis on a 1.5% agarose gel for about 1 h, and the characteristic 389 - bp fragments in the gel were visualized and photographed under the UV light. [Conclusion]The sensitivity of this methods was up to 40-50cfu/ml.To improve the specificity of the reaction, the amounts of the Mg2+ ,the primers and the template used were also optimized.