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目的基于SYBR Green I染料建立沙门菌、志贺菌和金黄色葡萄球菌的多重荧光定量PCR检测方法。方法针对沙门菌invA基因、志贺菌ipaH基因和virA基因、金黄色葡萄球菌nuc基因和femA基因片段设计引物,根据Tm值的差异分析确定荧光定量PCR扩增的靶基因和特异性引物,通过优化PCR反应体系,建立了用SYBR GreenⅠ多重荧光定量PCR熔解曲线来检测多种致病菌的方法。结果该方法检测的菌液灵敏度分别是沙门菌168 CFU/ml,志贺菌136CFU/ml,金黄色葡萄球菌1240CFU/ml,特异性强,整个过程只需要2h。结论该方法能快速、灵敏、特异检出沙门菌、志贺菌和金黄色葡萄球菌。
Objective To establish a multiplex fluorescence quantitative PCR method for the detection of Salmonella, Shigella and Staphylococcus aureus based on SYBR Green I dye. Methods Primers were designed according to the invA gene of Salmonella, ipaH gene and virA gene of Shigella, nuc gene and femA gene of Staphylococcus aureus, and the target genes and specific primers amplified by fluorescence quantitative PCR The PCR reaction system was optimized, and the method of using SYBR Green Ⅰ multiple fluorescence quantitative PCR melting curve to detect a variety of pathogenic bacteria was established. Results The sensitivities of this method were: Salmonella 168 CFU / ml, Shigella 136CFU / ml, Staphylococcus aureus 1240CFU / ml, the specificity was high and the whole process only took 2 hours. Conclusion The method can detect Salmonella, Shigella and Staphylococcus aureus quickly and sensitively.