建立Taqman探针实时荧光PCR检测食品中肠炎沙门氏菌的方法

目的利用Taqman探针实时荧光PCR检测食品中肠炎沙门氏菌。方法根据沙门氏菌属共有基因序列和肠炎沙门氏菌的特异序列分别设计2组引物及探针,运用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果采用沙门氏菌属探针可检测到25种不同血清型沙门氏菌(共49株),而11株阴性对照菌株未得到扩增;利用肠炎沙门氏菌探针可从25种不同血清型的沙门氏菌(共49株)和11株阴性对照菌株中特异性的检测出全部15株肠炎沙门氏菌;以肠炎沙门氏菌系列稀释度菌液DNA为模板进行实时荧光PCR扩增,菌株的模板浓度与Ct值之间呈现良好线性关系,线性系数(R2)0.993,扩增效率87%,最低检测浓度260cfu/mL;分别接种肠炎沙门氏菌于四种样品进行模拟样品检测,实时荧光PCR检测结果与经典培养鉴定方法的检测结果相吻合。结论实时荧光PCR检测食品中肠炎沙门氏菌的方法特异性强、敏感度高,可应用于食品中肠炎沙门氏菌的检测。

The detection of Salmonella Enteritidis with Taqman probe by real-time fluorescent PCR in food industry

Objective An assay was developed for detecting Salmonella Enteritidis in food with Taqman probe by real-time fluorescent PCR.Methods According to the shared gene sequences of Salmonella and the specific sequences of Salmonella Enteritidis,two sets of primers and probe were designed to conduct specific,sensitive and simulation sample tests by real-time PCR.Results The former set of primers and probe correctly detected 25 different Salmonella serotypes(49 strains),whereas 11 non-Salmonella strains didn’t present any amplification.The latter set of primers and probe successfully distinguished 15 Salmonella Enteritidis from 25 different Salmonella serotypes and other 11 non-Salmonella strains.Gradient dilutions of Salmonella Enteritidis were used as template to conduct real-time PCR test which presented linear relationship between the concentration of template and Ct value.Linear coefficient(R2),efficiency and detection limit were 0.993,87% and 260 cfu/mL respectively.4 kinds of simulation samples inoculated with Salmonella Enteritidis were detected by real-time PCR.The PCR method yielded a 100% correlation with results obtained by a conventional culture method.Conclusion The real-time PCR described here showed a high selectivity and sensibility and it could be a method for detecting Salmonella Enteritidis in food industry.