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Direct detection of methicillin-resistant in Staphylococcus spp. in positive blood culture by isothermal recombinase polymerase amplification combined with lateral flow dipstick assay
World Journal of Microbiology and Biotechnology ( IF 4.1 ) Pub Date : 2020-09-29 , DOI: 10.1007/s11274-020-02938-8
Arpasiri Srisrattakarn , Patcharaporn Tippayawat , Aroonwadee Chanawong , Ratree Tavichakorntrakool , Jureerut Daduang , Lumyai Wonglakorn , Pirom Sooksongsoontorn , Aroonlug Lulitanond

Methicillin-resistant staphylococci (MRS) are important antimicrobial-resistant pathogens in sepsis. Conventional blood cultures take 24-72 h. The polymerase chain reaction (PCR)-based methods give faster results (2-3 h) but need expensive thermal cyclers. We therefore developed an isothermal recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay for rapid detection of MRS in spiked blood culture samples. Fifty-six clinical isolates including 38 mecA-carrying staphylococci and 18 non-mecA-carrying organisms as confirmed by PCR methods were studied. RPA primer set and probe specific for mecA gene (encoding penicillin-binding protein 2a) were designed. RPA reaction was carried out under isothermal condition (45 °C) within 20 min and read by LFD in 5 min. The RPA-LFD provided 92.1% (35/38) sensitivity for identifying MRS in positive blood culture samples, and no cross-amplification was found (100% specificity). This test failed to detect three mecA-carrying S.sciuri isolates. The detection limits of RPA-LFD method for identifying MRS were equal to those of PCR method. The RPA-LFD is simple, fast, and user-friendly. This method could detect the mecA gene directly from the positive blood culture samples without requirement for special equipment. This method would be useful for appropriate antibiotic therapy and infection control, particularly in a low-resource setting.

中文翻译:

直接检测葡萄球菌中的耐甲氧西林。等温重组酶聚合酶扩增结合侧流试纸法检测阳性血培养

耐甲氧西林葡萄球菌 (MRS) 是脓毒症中重要的抗微生物病原体。传统的血培养需要 24-72 小时。基于聚合酶链反应 (PCR) 的方法可提供更快的结果(2-3 小时),但需要昂贵的热循环仪。因此,我们开发了一种等温重组酶聚合酶扩增 (RPA) 与横向流动试纸 (LFD) 检测相结合的方法,用于快速检测加标血培养样品中的 MRS。研究了 56 个临床分离株,包括 38 个携带 mecA 的葡萄球菌和 18 个非携带 mecA 的微生物,经 PCR 方法证实。设计了特异于 mecA 基因(编码青霉素结合蛋白 2a)的 RPA 引物组和探针。RPA 反应在等温条件 (45°C) 下在 20 分钟内进行,并在 5 分钟内通过 LFD 读取。RPA-LFD 提供了 92。在阳性血培养样本中识别 MRS 的灵敏度为 1% (35/38),未发现交叉扩增(100% 特异性)。该测试未能检测到三个携带 mecA 的 S.sciuri 分离株。RPA-LFD法检测MRS的检出限与PCR法相同。RPA-LFD 简单、快速且用户友好。该方法可直接从阳性血培养样本中检测mecA基因,无需特殊设备。这种方法可用于适当的抗生素治疗和感染控制,特别是在资源匮乏的环境中。RPA-LFD 简单、快速且用户友好。该方法可直接从阳性血培养样本中检测mecA基因,无需特殊设备。这种方法可用于适当的抗生素治疗和感染控制,特别是在资源匮乏的环境中。RPA-LFD 简单、快速且用户友好。该方法可直接从阳性血培养样本中检测mecA基因,无需特殊设备。这种方法可用于适当的抗生素治疗和感染控制,特别是在资源匮乏的环境中。
更新日期:2020-09-29
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