食源性MRSA双色荧光PCR检测方法建立

Establishment of double-colored real-time fluorescence PCR methods for detection of foodborne methicillin-resistant Staphylococcus aureus

中文关键词:  食源性  耐甲氧西林金黄色葡萄球菌  双色荧光PCR  检测

英文关键词:foodborne  methicillin-resistant Staphylococcus aureus  double-colored real-time fluorescence PCR  detection

中文摘要:

      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mecA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mecA基因的灵敏度可达2.7×103 CFU/mL, 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。

英文摘要:

      Objective To establish a double-colored real-time fluorescence PCR method of detection of foodborne Staphylococcus aureus by Taqman probe. Methods Two pairs of specific primers and Taqman fluorescent probe were designed and synthesized according to nuc gene for species identification of S.aureus and mecA gene for determination gene of MRSA. Double-colored real-time fluorescence PCR amplification system was developed. The specificity and sensitivity were tested by the established method. In order to validate the stability of method, strains of different generations were tested, after strains of S.aureus were subcultured successively. The feasibility and practicality were studied by detecting isolates from practical samples. Results MRSA and MSSA could be detected accurately, rapidly and specifically in this study. The sensitivity of detec-tion method was 2.7×103 CFU/mL for nuc and mecA gene of MRSA. The detection results of different generations were consistent. Conclusion This method has high specificity, sensitivity and stability for double-colored real-time fluorescence PCR method, and it can be applied to rapid detection and identification of foodborne MRSA.