摘要
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300cfu/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。
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 Salmonella 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(R 2),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.
出处
《食品安全质量检测学报》
CAS
2012年第4期300-305,共6页
Journal of Food Safety and Quality
基金
辽宁省自然科学基金项目(20102080)
