Taqman MGB PCR定量检测水产品中沙门氏菌的方法建立

建立采用Taqman MGB实时荧光PCR法快速定量检测水产品中沙门氏菌。根据沙门氏菌fimY基因保守序列，设计引物和Taqman MGB探针，建立Taqman MGB实时PCR定量检测体系。采用本方法对24株共14种血清型沙门氏菌和17株与沙门氏菌亲缘关系比较近，以及在样品中能同时存在的常见食源性致病菌菌株进行PCR扩增。结果显示：所有的沙门氏菌菌株结果均为阳性，而非沙门氏菌菌株检测结果均为阴性，反应特异性为100%。本方法的纯菌最低检测低限为13CFU/ml，样品江瑶贝和蚬子肉中添加肠炎沙门氏菌的最低检测低限为130CFU/ml；香螺肉中添加肠炎沙门氏菌的最低检测低限为1300CFU/ml。定量关系式为y=－3.381418lnx+45.115715，R2= 0.964878。整个实验2h即可完成，可应用于水产品中沙门氏菌污染状况调查及快速检测。

Real-time Fluorescent Quantitative PCR Assay of Salmonella in Seafood Using Taqman MGB Probe

A real-time fluorescent PCR array using the optimal combination of primer and Taqman MGB probe designed from the fimY conserved sequence of Salmonella was developed for the rapid and quantitative detection of Salmonella in seafood. Totally 24 strains of 14 serotypes of Salmonella and its 17 close relatives as well as common foodborne pathogenic bacteria coexisting in a sample were subjected to detection using the method. Results were positive for all Salmonella strains and negative for all other species tested with a specificity of 100%. The sensitivity of the method was sufficient to detect 13 CFU/ml of Salmonella in pure cultures, 130 CFU/ml of S.enteritidis spiked in shell ligament or shigimi meat, and 1300 CFU/ml of S.enteritidis spiked in moon snail meat. This method exhibited a quantification regression equation of y = －3.381418ln(x)＋45.115715, R2 = 0.964878, and could be completed within 2 h, thereby providing a useful approach for the rapid detection of Salmonella in seafood without prior isolation and characterization of strains which are needed in traditional microbiological methods.