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目的建立快速检测食源性耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)Taqman探针双色荧光PCR方法。方法根据金黄色葡萄球菌种属鉴定nuc基因和MRSA决定因子mec A基因,设计合成引物探针,建立双色荧光PCR扩增体系。利用所建立的方法检测特异性及灵敏度。将金黄色葡萄球菌依次传代培养,检测不同代次的菌株验证方法的稳定性,并对实际样品分离株进行检测验证方法的可行性与实用性。结果该方法可准确并特异性检测出MRSA和甲氧西林敏感金黄色葡萄球菌(methicillin-susceptible Staphylococcus aureus,MSSA),检测MRSA的nuc基因和mec A基因的灵敏度可达2.7×103 CFU/m L,不同代次的菌株的检测结果一致。结论本实验所建立的双色荧光PCR检测方法具有良好的特异性、灵敏度及稳定性,可用于快速检测食源性MRSA。
Objective To establish a dual-color fluorescent PCR method for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) Taqman probe. Methods According to the genera of Staphylococcus aureus, the nuc gene and MRSA determinant mec A gene were designed and synthesized. Primer probes were designed and synthesized. Two-color fluorescent PCR amplification system was established. The established method was used to detect the specificity and sensitivity. The Staphylococcus aureus was subcultured in succession to test the stability of different generations of strains to verify the method. The feasibility and practicability of the method were tested and validated. Results The method could accurately and specifically detect MRSA and methicillin-susceptible Staphylococcus aureus (MSSA). The sensitivity of nuc gene and mec A gene in MRSA was 2.7 × 103 CFU / m L , Different generations of strains of the same test results. Conclusion The two-color fluorescence PCR assay established in this experiment has good specificity, sensitivity and stability and can be used for rapid detection of food-borne MRSA.