摘要

基因mecA及其同系物mecC在金黄色葡萄球菌和其他葡萄球菌中赋予甲氧西林抗性。耐甲氧西林葡萄球菌（MRS）被认为对所有β-内酰胺类抗生素耐药。为了避免使用β-内酰胺类抗生素控制MRS感染，迫切需要对mecA和mecC进行快速可靠的筛查检测，这些检测可以很容易地整合到常规实验室诊断中。本研究的目的是基于核酸横向流动免疫分析（NALFIA）技术开发这种快速检测耐甲氧西林方法。在NALFIA中，靶序列经PCR扩增，通过抗原 - 抗体相互作用固定化，并最终通过生物素 - 中性亲和素相互作用显现为由neutravidin标记的碳颗粒产生的独特黑条。使用PCR-NALFIA对临床样品中60种确定的菌株（MRS和非目标细菌）和28种耐甲氧西林金黄色葡萄球菌（MRSA）分离株进行筛选，与PCR进行后续凝胶电泳（PCR-GE）时PCR。虽然所有的样品都能正确识别所有的检测方法，但PCR-NALFIA在检测限上更为优越。此外，该测定通过在NALFIA测试条上的不同位置处显现两个等位基因而允许mecA和mecC之间的区分。然而，由于该测试系统仅针对mecA和mecC基因，因此不能确定mec基因在哪些葡萄球菌种类中被包括。只需要文化方法所需时间的一小部分（即黄金标准），这里介绍的PCR-NALFIA易于处理并且可以很容易地整合到实验室诊断中。

The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all β-lactam antibiotics. To avoid the use of β-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA andmecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.

https://www.sciencedirect.com/science/article/pii/S0378113516302243