细菌超广谱β-内酰胺酶耐药基因多重实时荧光PCR检测体系的建立

Establishment of Multiplex Real-time Fluorescent PCR Detection System for Bacterial Extended-spectrumβ-lactamase Resistance Gene

目的建立细菌超广谱β-内酰胺酶(extended spectrum beta-lactamases,ESBLs)耐药基因多重实时荧光聚合酶链式反应(polymerase chain reaction,PCR)法,测定其检测的敏感性、特异性及灵敏度。方法选取70例经普通PCR和DNA测序明确其超广谱β-内酰胺酶耐药基因类型的菌株,建立改良实时荧光PCR体系,确定细菌超广谱β-内酰胺酶耐药基因blaTEM、blaSHV、blaOXA-1、blaCTX-M-1和blaCTX-M-9熔解曲线的Tm值。分别以普通多重PCR和多重实时荧光PCR,检测受检菌株中blaTEM、blaSHV、blaOXA-1、blaCTX-M-1和blaCTX-M-9基因的存在情况,对比两组中不同基因检出的阳性率,计算敏感性和特异性。选定用于灵敏度检测的菌株,以活菌计数的方式稀释细菌菌液,分别进行普通多重PCR和多重实时荧光PCR扩增,以电泳出现目的条带为阳性判定标准,将目的条带即将消失的相应浓度作为检测方法的灵敏度。结果多重实时荧光PCR法中,blaTEM、blaSHV、blaOXA-1、blaCTX-M-1和blaCTX-M-9基因的熔解曲线Tm值分别是77.5℃、94.7℃、82.8℃、91.6℃、92.6℃;这五种基因联合检测时,能同时检测出blaSHV、blaCTX-M-1、blaCTX-M-9三种基因。多重实时荧光PCR对blaSHV、blaCTX-M-1、blaCTX-M-9基因检测的特异性和敏感性均为100%,灵敏度是102 cfu/μL。结论建立了用于检测超广谱β-内酰胺酶耐药基因的多重实时荧光PCR法,与普通多重PCR相比,具有较高的灵敏度。

Objective To establish a multiplex real-time polymerase chain reaction(PCR)method for bacterial extended spectrum beta-lactamases(ESBLs)resistance gene and determine its specificity and sensitivity.Methods Seventy strains with extended-spectrumβ-lactamase resistance gene types were identified by ordinary PCR and DNA sequencing.An improved real-time PCR system was established to determine Tm values of the bacterial extended-spectrumβ-lactamase resistance gene blaTEM,blaSHV,blaOXA-1,blaCTX-M-1 and blaCTX-M-9.The blaTEM,blaSHV,blaOXA-1,blaCTX-M-1 and blaCTX-M-9 genes in the strains were detected by common multiplex PCR and real-time PCR,and the positive rates of different genes detected in the two groups were compared.Calculated sensitivity and specificity.The strains selected for sensitivity detection were diluted with the bacteria count,and the common multiplex PCR and real-time PCR amplification were performed respectively.The target band was positively determined by electrophoresis,and the target band was about to disappear.The corresponding concentration was used as the sensitivity of the detection method.Results In the multiplex real-time PCR method,the Tm values of the blaTEM,blaSHV,blaOXA-1,blaCTX-M-1 and blaCTX-M-9 genes were 77.5°C,94.7°C,82.8°C,91.6°C,and 92.6°C,respectively.When these five genes are combined,the three genes blaSHV,blaCTX-M-1,blaCTX-M-9 can be detected simultaneously.The specificity and sensitivity of multiplex blaSHV,blaCTX-M-1,blaCTX-M-9 genes were 100%,and the sensitivity was 102 cfu/μL.Conclusion The multiplex real-time PCR method for the detection of extended-spectrumβ-lactamase resistance genes was established,which has higher sensitivity compared with the normal multiplex PCR.