建立实时荧光PCR快速鉴定鸡制品中鸡伤寒沙门氏菌的方法

The rapid identification method of Salmonella gallinarumby real-time fluorescent PCR

建立基于Taqman探针实时荧光PCR检测鸡伤寒沙门氏菌(Salmonella gallinarum)的方法.根据GenBank公布的鸡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34株鸡伤寒沙门氏菌菌株、26株其他血清型沙门氏菌和7株非沙门氏菌菌株中检测出全部的34株鸡伤寒沙门氏菌.以鸡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0.999,扩增效率为88.2%,最低检测浓度为5cfu/mL.对已接种鸡伤寒沙门氏菌的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.该方法特异性好、灵敏度高,是快速鉴定鸡伤寒沙门氏菌的有效方法.

A method was developed for rapid identification of detecting Salmonella gallinarum by real-time fluorescent PCR .According to the gene of Genbank ,a set of primers and Taqman probe was de-signed to perform specific ,sensitive and simulation sample tests by real-time PCR .The results were confirmed that the specificity probe correctly distinguished 34 Salmonella gallinarum strains from 24 other Salmonella serotypes strains and 7 non-Salmonella strains .Gradient dilutions of Salmonella gallinarum 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 .999 ,88 .2% and 5 cfu/mL correspondingly ,simulation samples inoculated with Salmo-nella gallinarum were detected by real-time PCR assay .The PCR method yielded a 100% correlation with results obtained by conventional culture method .The newly method showed that a high specific-ity ,sensibility and accuracy could be applied for rapid identification of Salmonella gallinarum .