Unité de Recherche sur Maladies Infectieuses et Tropicales Emergents, CNRS-IRD, UMR 6236, Faculté de Médecine et de Pharmacie, Université de Méditerranée Aix-Marseille II, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France.
Int J Antimicrob Agents. 2011 Jun;37(6):544-6. doi: 10.1016/j.ijantimicag.2011.02.006. Epub 2011 Apr 14.
In this study, we report the development of a rapid real-time polymerase chain reaction (PCR) assay with TaqMan probe to detect the New Delhi metallo-β-lactamase (NDM-1)-encoding gene directly from bacterial isolates. The specificity of the assay was verified in silico as well as with a large panel of 84 clinically relevant bacteria, including the Klebsiella pneumoniae NCTC 13443 NDM-1-positive reference strain. Using this assay retrospectively on a local series of 44 K. pneumoniae isolates from Marseille Hospitals (France), it was possible to detect and identify an NDM-1-producing K. pneumoniae strain isolated from bronchoalveolar lavage in April 2010 from a French patient repatriated from India after a motorbike accident. Standard PCR amplification and sequencing of the entire NDM-1 gene from this isolate was also performed and the amino acid sequence showed 100% homology with the NDM-1 protein from the K. pneumoniae reference strain. We believe that this real-time PCR assay would be a powerful tool that could be added to other molecular detection assays such as detection of KPC- or OXA-encoding genes for rapid screening and/or identification of carbapenem-resistant bacterial isolates from patients returning from the Asian continent that could be implemented in a point-of-care strategy.
在这项研究中，我们报告了一种快速实时聚合酶链反应（PCR）检测方法的开发，该方法使用 TaqMan 探针直接从细菌分离物中检测新德里金属β-内酰胺酶（NDM-1）编码基因。该检测方法的特异性已通过计算机模拟以及 84 种临床相关细菌的大型面板进行了验证，其中包括产 NDM-1 的肺炎克雷伯菌 NCTC 13443 参考菌株。使用该检测方法回顾性地对来自马赛医院（法国）的 44 株肺炎克雷伯菌进行检测，我们能够检测并鉴定出一株产 NDM-1 的肺炎克雷伯菌菌株，该菌株于 2010 年 4 月从一名在印度因摩托车事故返回的法国患者的支气管肺泡灌洗液中分离出来。还对该分离物的整个 NDM-1 基因进行了标准 PCR 扩增和测序，该氨基酸序列与肺炎克雷伯菌参考菌株的 NDM-1 蛋白完全同源。我们认为，这种实时 PCR 检测方法将是一种强大的工具，可以与其他分子检测方法（如检测 KPC-或 OXA 编码基因）结合使用，用于快速筛选和/或鉴定从亚洲大陆返回的耐碳青霉烯类细菌分离物，可在即时护理策略中实施。
