食品中伤寒沙门氏菌TaqMan探针实时PCR检测方法

The Rapid Identifi cation Method of Salmonella Typhi with Taqman Probe Real-Time Fluorescent PCR

建立实时荧光PCR快速鉴定伤寒沙门氏菌(Salmonella Typhi)的方法。根据GenBank公布的伤寒沙门氏菌基因序列,设计引物和Taqman探针,采用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果表明,特异性引物和探针可从31株伤寒沙门氏菌菌株、27株其他血清型沙门氏菌和7株非沙门氏菌菌株中鉴定出全部的31株伤寒沙门氏菌。以伤寒沙门氏菌梯度稀释菌液DNA为模板进行实时荧光PCR扩增,菌株模板浓度与Ct值呈良好线性关系,线性系数为0.994,扩增效率为94.5%,最低检测浓度为4cfu/mL的添加浓度。实时荧光PCR检测与传统方法相比较,两者结果一致。该方法特异性好、灵敏度高,可以快速鉴定伤寒沙门氏菌。

A method was developed for Rapid Identification of Salmonella Typhi with real-time fluorescent PCR. According to the gene of Genbank, a set of primers and Taqman probe was designed to perform specific, sensitive and simulation sample tests with real-time PCR. The results showedthe specificity probe correctly distinguished 31 Salmonella Typhi strains from 27 other Salmonella serotypes strains and 7 non-Salmonella strains. Gradient dilutions of Salmonella Typhi were used as template to perform real-time PCR assay which presented linear relationship between the concentration of template and Ct value. Linear coefficient （R2）, efficiency and detection limit were 0.994, 94.5%and 4cfu/ml correspondingly. Simulation samples inoculated with Salmonella Typhi were detected with real-time PCR assay. The PCR method yielded a 100%correlation with results obtained by conventional culture method. The new method that showed a high specificity, sensibility and accuracy could be applied for the rapid identification of Salmonella Typhi.