多重PCR检测食源金黄色葡萄球菌nuc、blaZ和mecA基因方法的建立与应用

Multiplex PCR for detection of nuc,blaZ and mecA genes in food-borne Staphylococcus aureus and its application

目的建立快速检测食源金黄色葡萄球菌(Sa)耐热核酸酶基因(nuc)、β-内酰胺酶基因(blaZ)和甲氧西林耐药基因(mecA)的多重PCR方法。方法根据GenBankSa的nuc、blaZ和mecA基因序列,设计3对特异性引物,建立鉴定Sa及分析Sa耐β-内酰胺类抗生素三重PCR方法 ,并对79株食源Sa进行基因检测与表型比较。结果显示供试Sa中检出nuc、blaZ基因阳性率分别为97.47%、73.42%,mecA基因检出为阴性;nuc基因检出与其表型符合率达97.47%;而blaZ扩增结果与β-内酰胺酶试验、青霉素类敏感试验同时符合率为49.37%,前者与后两者符合率分别为60.76%和75.95%;mecA与耐甲氧西林表型符合率为100%;此次79株食源Sa中无耐甲氧西林Sa。结论该方法简便、快捷、准确,为食源Sa鉴定和耐药性分子分析提供了参考依据。

According to nuc,blaZ and mecA gene sequences of Staphylococcus aureus （SA） in GenBank,three pairs of specific primers had been designed and used in triple PCR for identification and analysis on SA beta-lactam resistant antibiotics. Multiplex PCR had been developed and optimized in the detection of 79 isolation strains in food-borne SA,and then the results were compared with phenotypes. The results indicated that the positive rates for detection of nuc and blaZ genes by PCR were 97.47% and 73.42% respectively,while PCR results of mecA gene was negative. The coincidence rate between nuc gene and phenotype detected by PCR method was 97.47%. The coincidence rate among blaZ gene detection,beta lactamase assay and penicillin sensitivity test was 49.37%. The coincidence rate between blaZ gene detection and beta lactamase assay was 60.76%,and the coincidence rate between blaZ gene detection and penicillin sensitivity test was 75.95%. The coincidence rate between mecA gene detection by PCR and methicillin resistant phenotype was 100%. There was no methicillin-resistant SA in the 79 strains of SA isolated from animal origin food. It＇s suggested that multiplex PCR assay was convenient,time-saving and accurate. Our study provides reference for identification of food-borne SA and molecular analysis of antimicrobial resistance in SA.