沙门氏菌和大肠杆菌O_(157):H_7双重荧光PCR检测方法的研究

Development of A Dual Real-Time PCR For the Detection of Salmonella spp and Escherichia Coli O_(157):H_7

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。

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 realtime 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：H7were 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.