荧光实时定量PCR检测溶藻弧菌方法的建立

目的 建立荧光实时定量PCR(qPCR)定量检测溶藻弧菌的方法,检测该方法的灵敏度并与传统细菌培养比较其特异性吻合率。方法 选择溶藻弧菌的鞭毛基因fliC为目的基因设计探针引物,建立Taqman探针qPCR体系。采用双盲法设计,分别用qPCR和Biolog Microstation System对溶藻弧菌和10株相关细菌进行鉴定,以考核qPCR检测体系的特异性。同时,通过梯度稀释和绘制标准曲线,评价利用qPCR检测溶藻弧菌的灵敏度。结果 本试验建立的qPCR方法能特异、准确、快速鉴定溶藻弧菌,敏感度达102CFU/ml。结论 qPCR方法能够有效快速检测溶藻弧菌。

The method of fluorescent real-time quantitative PCR for detecting alginolyticus

Objective To establish a fluorescent real-time quantitative PCR method (qPCR) to detect alginolyticus,to evaluate the sensitivity of this technique and compare the specificity with traditional germiculture method.Methods The flagella gene (fliC) of alginolyticus was selected as a target gene to design probe primer and establish Taqman probe qPCR system.In order to test the specificity of qPCR system,a double-blind design was adapted to identify alginolyticus and other relating bacteria with qPCR and Biolog Microstation System.At the same time,the methods of stepped attenuation and standard curve were used to test the sensitivity.Results The qPCR method had the ability to identify alginolyticus specifically,sensitively and rapidly.The sensitivity reached to 102 cfu/ml.Conclusion qPCR can detect alginolyticus effectively and rapidly.