Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, PantonValentine leucocidin (PVL), mecA and homologue mecALGA251
Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, PantonValentine leucocidin (PVL), mecA and homologue mecALGA251
复制标题
DOI：
10.1093/jac/dks221
复制
发表时间：
2012-10-01
影响因子：
5.2
通讯作者：
Kearns, Angela M.
中科院分区：
文献类型：
--
作者：
Pichon, Bruno;Hill, Robert;Kearns, Angela M.
最近发现的 mecA 同源物 (mecA(LGA251)) 在两个基因变体之间具有高度变异性，这表明在没有抗菌药物敏感性测试的情况下，携带 mecA(LGA251) 的金黄色葡萄球菌可能被错误地识别为甲氧西林敏感金黄色葡萄球菌 (MSSA)。 四重 PCR 测定可明确区分 mecA 和 mecA(LGA251)，以及 nuc 基因（物种特异性标记）和 lukS-PV 基因 [编码 PantonValentine 杀白细胞素 (PVL) 毒素] 的检测。使用 (i) PVL 阳性和 PVL 阴性 MSSA 和 耐甲氧西林金黄色葡萄球菌 (MRSA) 和 (ii) 来自英国、丹麦和法国的已知含有 mecA(LGA251) 的 MRSA。当应用于对苯唑西林不敏感、mecA 阴性的人类分离株进行回顾性收集时，发现三个编码 mecA(LGA251)，其中一个来自血液，这是迄今为止在英格兰确认的第一例因具有 mecA(LGA251) 的金黄色葡萄球菌引起的菌血症病例。最后，该检测于 2011 年 8 月被引入常规葡萄球菌参考单位（HPA 微生物学服务，英国伦敦）工作流程，并且在使用的前 5 个月内，从测试的 2263 个金黄色葡萄球菌中鉴定出 10 个含有 mecA 同源物的分离株，这表明英格兰人群中的循环较低但持续。
The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing.In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the PantonValentine leucocidin (PVL) toxin].The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.