食源性MRSA氨基糖苷类耐药基因多重PCR方法建立

战晓微; 郑秋月; 傅俊范; 徐君怡; 陈璐; 董姝君

doi:10.13386/j.issn1002-0306.2015.01.055

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

摘要

摘要: 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。

Abstract: 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,mec A for MRSA,aac A- aph D and aph( 3')- Ⅲa 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.72 strains of S.aureus all had nuc,the coincidence rate of mec A and MRSA was 100%,the coincidence rate of aac A- aph D,aph( 3')- Ⅲa 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.