金黄色葡萄球菌肠毒素快速分型体系的建立及应用

Establish and apply a rapid detection system of Staphylococcus aureus enterotoxin genes

目的建立SYBR-Green荧光PCR体系金黄色葡萄球菌肠毒素快速分型体系,应用于金黄色葡萄球菌食物中毒和食品风险监测快速诊断。方法根据GenBank公布的金黄色葡萄球菌肠毒素A、B、C、D、E基因的保守序列设计特异性引物,分别建立SYBR-Green荧光PCR体系。应用该系列体系检测185株金黄色葡萄球菌野生株的肠毒素基因A～E型。结果建立的SYBR-Green荧光PCR系列体系其DNA灵敏度为1.34～2.80ng/μL,菌液灵敏度为46～96CFU/mL;用以检测185株金黄色葡萄球菌的肠毒素基因A～E型,携带一种基因型的为58.38%,同时携带两种以上毒素基因占4.86%。结论建立的荧光PCR反应体系快速、灵敏度高、特异性强,能应用于金黄色葡萄球菌肠毒素基因的准确分型,对食物中毒诊断和食品风险监测很有意义。

Objective To establish a new rapid and highly sensitive detection system for Staphylococcus aureus（S.aureus） enterotoxin genes using SYBR green real-time PCR and its application of rapid diagnosis for alimentary toxicosis caused by S.aureus and related food risks.Methods Primers specific for S.aureus enterotoxin A,B,C,D and E genes were designed according to GenBank data.PCR systems based on SYBR-Green were constructed for each gene and performed for the detection of A-E genes in 185 wild strains of S.aureus.Results For the real time PCR assay,the DNA sensitivity achieved 1.34-2.80 ng/μL and the bacterial solution sensitivity was 46-96 CFU/mL.58.38% of the 185 wild strains of S.aureus were with single gene type of A-E genes,and 4.86% were with tow type of toxin genes,when being detected by the constructed PCR systems.Conclusion A rapid PCR system with high sensitivity and specificity was successfully established,and could be used for the accurate typing of S.aureus entertoxin genes,for the diagnosis of alimentary toxicosis and for the monitoring of food risks.