Choi Ji Ae, Bae Song Mee, Kim Jung Wook, Lee Kwang Jun
Division of Antimicrobial Resistance, Center for Infectious Diseases Research, National Institute of Health, Korea Centers for Disease Control and Prevention, Cheongju, Korea.
Osong Public Health Res Perspect. 2020 Feb;11(1):53-59. doi: 10.24171/j.phrp.2020.11.1.08.
Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.
The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing . It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.
No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.
The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.
碳青霉烯类耐药是一种严重的临床和公共卫生威胁。碳青霉烯酶可导致碳青霉烯类耐药，且大多数碳青霉烯酶基因由质粒编码，因此耐药性很容易传播。在本研究中，我们旨在开发一种基于TaqMan平台的新型系统，用于快速检测6种临床常见的碳青霉烯酶基因：碳青霉烯酶、新德里金属β-内酰胺酶、氧青霉烷酶、亚胺培南水解酶、维罗纳整合子编码金属β-内酰胺酶和圭亚那超广谱β-内酰胺酶。
通过检测6株产碳青霉烯酶菌株的基因组DNA对三重检测法进行验证。通过直接菌落三重实时聚合酶链反应（PCR），用310株肠杆菌科分离株组成的盲法样本进行验证，其中包括225株产碳青霉烯酶菌株和85株非产碳青霉烯酶菌株。使用ABI 7500快速仪器（美国加利福尼亚州应用生物系统公司）进行实时PCR，并设计针对每个碳青霉烯酶靶点的特异性引物，其中包括修饰的肽核酸寡核苷酸。
在阴性样本中未检测到扩增。结果显示与先前鉴定的基因型100%一致。整个检测，包括DNA提取和实时PCR，在2小时内完成。
新开发的三重实时PCR检测法可用于快速、准确且同时检测肠杆菌科中的6种碳青霉烯酶基因，表明其有潜力为医院中耐药感染的适当治疗、管理和传播预防提供早期决策依据。
