沙门氏菌和大肠杆菌O157:H7双重荧光PCR检测方法的研究

目的建立一种能同时检测沙门氏菌和大肠杆菌O157:H7的双重荧光PCR方法,应用于动物源性食品的快速检验。方法根据沙门氏菌invA基因和大肠杆菌O157:H7 RFBE基因的保守序列,设计引物和探针,通过优化反应条件,建立可同时检测沙门氏菌和大肠杆菌O157:H7的双重荧光PCR方法,应用于动物源性食品的检验,并与miniVIDAS快速初筛方法和SN标准方法进行比较。结果本研究建立的双重荧光PCR方法可同时快速检测沙门氏菌和大肠杆菌O157:H7,对纯菌的检测灵敏度均低于10CFU/双重荧光PCR反应体系。应用本方法检测36株标准/参考菌株,结果只有9株目的菌标准/参考菌株出现特异性扩增,其余27株非目的菌均呈阴性反应。定量检测重复性试验结果,批内和批间的变异系数均小于2%。应用本方法检测人工染菌样品,结果与miniVIDAS和SN方法检测结果一致,但检测时间比miniVIDAS快了3倍,比SN标准快了10多倍。结论本研究建立的双重荧光PCR方法具有快速、灵敏、特异、重复性好的优点,可在8小时内完成样品沙门氏菌和大肠杆菌O157:H7的检验。

Development of A Dual Real-Time PCR For the Detection of Salmonella spp and Escherichia Coli O157:H7

Objective To develop a dual real-time PCR assay for the simultaneous detection of Salmonella spp and Escherichia Coli O157:H7 in animal-origined food.Methods A dual real-time PCR was developed for the rapid detection of Salmonella spp and Escherichia Coli O157:H7 based on the primers and probes which were designed for the conservative domain of invA gene of Salmonella spp and RFBE gene of Escherichia Coli O157:H7.The dual real-time PCR assay was applied to samples artificially contaminated by Salmonella spp and Escherichia Coli O157:H7,which results were compared with those of miniVIDAS and SN standard.Results The dual real-time PCR developed can detect less than 10cfu/dual real-time PCR reaction both for Salmonella spp and Escherichia Coli O157:H7 with pure cultured broth.In the specificity testing,among 36 reference/standard strains,only target strains of Salmonella spp and Escherichia Coli O157:H7 were detected positively,while 27 non-target strains were all detected negatively.All of the coefficient of variation of intra-assay and inter-assay for quantitative detection were less than 2%.The results of dual real-time PCR detecting samples artificially contaminated are consistent with those of miniVIDAS and SN standard,but the dual real-time PCR is 3 times and more than 10 times faster than miniVIDAS and SN standard seperatively.Conclusions The dual real-time PCR is a repeatable,specific ,sensitive and rapid method for the detection of Salmonella spp and Escherichia Coli O157:H7.It takes less than 8h to qualitatively or quantitatively detect Salmonella spp and Escherichia Coli O157:H7 in samples.