多重降落PCR检测血流感染产ESBLs肠杆菌科细菌与MRSA方法的建立和应用  

Establishment and application of multiplex touchdown PCR for detection of extended-spectrum β-lactamases-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus in bloodstream infection

作　　者：毕艳妮[1] 隋振耿 宋宇[1] 曲业敏[1] 马淑青[1] 孙梅[1] 刘海珠[1] 

机构地区：[1]大连医科大学附属威海市立医院,山东威海264200

出　　处：《中国感染控制杂志》2015年第10期663-667,共5页Chinese Journal of Infection Control

摘　　要：目的探索一种可同时检测产超广谱β-内酰胺酶(ESBLs)的肠杆菌科细菌与耐甲氧西林金黄色葡萄球菌(MRSA)的多重降落PCR方法。方法收集某院20l3年3月—2014年9月102份住院患者血培养阳性标本,针对产ESBLs肠杆菌科细菌的SHV、TEM、OXA基因和MRSA的MecA基因设计4对特异性引物,分别采用单一降落PCR和多重降落PCR对各耐药基因进行扩增,并将结果与药敏纸片法进行比较。结果各单一PCR均可扩增出特异性条带,并用多重降落PCR同时检测了4种耐药基因;建立的多重降落PCR对4种耐药基因(MecA、SHV、TEM、OXA)DNA的检测下限分别为4.37、2.19、4.53和3.59 ng。与药敏纸片法相比,多重降落PCR总灵敏度与特异性分别为100.00%、88.24%,检测ESBLs的灵敏度达100.00%,特异性为87.23%,检测MRSA的灵敏度和特异度均为100.00%。结论该实验建立的多重降落PCR具有高灵敏度与特异性,可用于产ESBLs肠杆菌科细菌和MRSA所致血流感染的快速诊断与流行病学调查,有利于指导临床使用抗菌药物。Objective To develop a multiplex touchdown PCR for simultaneous detection of extended-spectrumβ-lactamases（ESBLs）-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus（MRSA）.Methods Blood culture positive specimens from102 hospitalized patients were collected between March 2013 and September 2014,four pairs of specific primers were designed based on SHV,TEM,and OXAgenes of ESBLs-producing Enterobacteriaceae and MecA gene of MRSA,drug-resistant genes were amplified with single touchdown PCR and multiplex touchdown PCR,the results were compared with Kirby-Bauer disk diffusion method.Results Each single PCR amplified a specific band,four drug-resistant genes were also detected by multiplex touchdown PCR;the lower detection limits of multiplex touchdown PCR for DNA of MecA,SHV,TEM,and OXA were 4.37 ng,2.19 ng,4.53 ng,and 3.59 ng,respectively.Compared with Kirby-Bauer disk diffusion method,the overall sensitivity and specificity of multiplex touchdown PCR were 100.00% and 88.24%respectively,for ESBLs were 100.00% and 87.23% respectively,for MRSA were both 100.00%.Conclusion A higher sensitivity and specificity multiple touchdown PCR assay has been developed,and it can be used in the rapid diagnosis and epidemiology investigation of bloodstream infection caused by ESBLs-producing Enterobacteriaceae and MRSA,and is helpful for guiding antimicrobial use in clinic.

关 键 词：超广谱β-内酰胺酶 ESBLS 耐甲氧西林金黄色葡萄球菌 MRSA SHV TEM OXA MECA 多重降落PCR 

分 类 号：R446.5[医药卫生—诊断学]