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

摘要

Objective To develop a multiplex touchdown PCR for simultaneous detection of extended-spectrum β-lactamases (ESBLs )-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA ). Methods Blood culture positive specimens from 102 hospitalized patients were collected between March 2013 and September 2014,four pairs of specific primers were designed based on SHV,TEM,and OXA genes of ESBLs-pro-ducing 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 ES-BLs 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 epidemi-ology investigation of bloodstream infection caused by ESBLs-producing Enterobacteriaceae and MRSA,and is help-ful for guiding antimicrobial use in clinic.%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。