Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Department of Medical Technology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
PLoS One. 2022 Jun 30;17(6):e0270686. doi: 10.1371/journal.pone.0270686. eCollection 2022.
Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.
金黄色葡萄球菌，特别是耐甲氧西林金黄色葡萄球菌（MRSA），是一种重要的细菌，可在全球范围内引起社区和与医疗保健相关的感染。然而，目前的常规检测方法耗时较长。因此，我们开发并评估了一种重组酶聚合酶扩增-侧流条带（RPA-LF）方法，用于检测阳性血培养物中的 MRSA。在进行扩增反应之前，直接对来自医院的 60 份阳性血培养物进行了测试，无需进行 DNA 提取和纯化。RPA 引物和探针针对 nuc（编码热核酸酶）和 mecA（编码青霉素结合蛋白 2a）基因进行设计，用于诊断金黄色葡萄球菌及其耐甲氧西林状态。RPA 反应在等温条件（45°C）下进行，20 分钟内完成，在室温下通过 LF 条带进一步 5 分钟提供结果。使用血培养物对 RPA-LF 的评估显示，RPA-LF 对鉴定金黄色葡萄球菌的敏感性为 93.3%（14/15），没有交叉扩增[特异性为 100%（45/45）]。对于检测耐甲氧西林，RPA-LF 检测的敏感性为 100%（16/16），特异性为 97.7%（43/44）。RPA-LF 快速、高度敏感、稳健且易于使用。它可以直接用于检测 MRSA，无需特殊设备。
