食源性MRSA氨基糖苷类耐药基因多重PCR方法建立

Establishment of Aminoglycoside resistance gene of foodborne MRSA detection method by Multiple- PCR

目的:了解食源性金黄色葡萄球菌中耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)及对氨基糖苷类庆大霉素、卡那霉素抗生素的耐药性情况,并建立快速检测鉴定金黄色葡萄球菌耐药性多重PCR方法。方法:对金黄色葡萄球菌进行增菌培养,并提取DNA作为模板,以nuc基因作为菌属鉴定基因,mec A基因作为MRSA检测基因,aac A-aph D及aph(3')-Ⅲa基因作为耐氨基糖苷类庆大霉素、卡那霉素的检测目的基因,建立四重PCR检测方法并进行特异性及灵敏度评价;利用所建立的方法检测72株食品及食物中毒患者来源的金黄色葡萄球菌。结果:成功建立四重PCR检测方法,且72株金黄色葡萄球菌均有nuc基因检出,mec A基因与MRSA检测符合率达100%,aac A-aph D、aph(3')-Ⅲa基因与庆大霉素、卡那霉素耐药菌株的符合率达95.12%。结论:本实验所建立的多重PCR方法灵敏度高、特异性好,可以快速检测出MRSA,并对氨基糖苷类庆大霉素、卡那霉素耐药菌株检测具有较高的准确率。

Objective： To investigate the prevalence of methicillin-resistant Staphylococcus aureus MRSA and resistance to Aminoglycoside gentamicin, kanamycin in foodborne S.aureus,and establish a rapid quadruple-PCR method for detection and identification antibiotic resistance of S.aureus.Methods： Genomic DNA was extracted as template for PCR from S.aureus recoveried and cultured in enrichment medium.According to nuc for Genus,mecA for MRSA, aacA-aphD and aph （3＇） - Ⅲ a for Gentamicin and kanamycin, quadruple- RCR detection method was developed, its sensitivity and specificity were evaluated; a total of 72 strains of S.aureus from food and patients with food poisoning were detected by quadruple RCR method. Results. Quadruple- RCR detection method was established.？2 strains of S.aureus all had nuc,the coincidence rate of mecA and MRSA was 100% ,the coincidence rate of aacA-aphD, aph （3＇） - Ⅲ a and gentamicin, kanamycin was 95.12%.Conclusion ： this established quadruple -RCR method was specific, sensitive. MRSA could be tested rapidly, and there was high accurate rate for detection of gentamicin,kanamycin resistant strains of S.aureus.