实时荧光PCR法检测鼠伤寒沙门氏菌

目的 建立实时荧光PCR法检测鼠伤寒沙门氏菌的方法。方法 基于鼠伤寒沙门氏菌II型限制酶基因, 设计引物及Taqman探针, 利用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果 特异性探针可从25种血清型沙门氏菌(共49株)及11株阴性对照菌株中检测出全部的11株鼠伤寒沙门氏菌。以鼠伤寒沙门氏菌梯度稀释菌液DNA为模板进行实时荧光PCR实验, 菌株模板浓度与Ct值呈良好线性关系, 线性系数(R2)为0.998, 扩增效率90%, 最低检测浓度300 cfu/mL。对已接种鼠伤寒沙门氏菌的4种模拟样品同时进行实时荧光PCR检测和传统方法鉴定, 两者结果一致。结论 此方法特异、灵敏、准确, 适于食品中鼠伤寒沙门氏菌的检测。

Detection of Salmonella typhimurium by real-time fluorescent PCR

Objective A method was developed for the detection of Salmonella typhimurium by real-time fluorescent PCR. Methods According to the gene of type II restriction enzyme, a set of primers and Taqman probe were designed to perform specificity, sensitivity and simulation sample tests by real-time PCR. Results The specificity probe correctly distinguished all of 11 Salmonella typhimurium strains from 25 kinds of Salmo-nella serotypes (49 strains) and 11 non-Salmonella strains. Gradient dilutions of Salmonella typhimurium were used as template to perform the real-time PCR assay, which presented a good linearity between the concentration of template and Ct value. Linear coefficient (R2), amplification efficiency and detection limit were 0.998, 90% and 300 cfu/mL, correspondingly. Four kinds of simulation samples inoculated with Salmonella typhimurium were detected by the real-time PCR assay. The results obtained by PCR method accorded with that obtained by conventional culture method. Conclusion The new method that showed a high specificity, sensibility and accuracy could be applied for the detection of Salmonella typhimurium in food.