金黄色葡萄球菌RAA-LFD快速检测方法的建立与应用

Development and Application of Recombinase-Aided Amplification Combined with Lateral Flow Dipstick Assay for Rapid Detection of Staphylococcus aureus

靶向金黄色葡萄球菌(Staphylococcus aureus)保守基因(nuc)序列设计特异性引物,经引物筛选和反应条件的优化,建立金黄色葡萄球菌的重组酶等温扩增(recombinase aided amplification,RAA)结合侧流层析试纸条(lateral flow dipstick,LFD)的快速检测方法。该方法在引物浓度200 nmol/L、33℃反应20 min即可实现靶标基因片段的有效扩增。特异性分析结果表明RAA-LFD方法检测金黄色葡萄球菌与其他常见致病菌菌株间不存在交叉反应,灵敏度分析结果表明RAA-LFD方法检测金黄色葡萄球菌的检出限为4 fg/μL(DNA)与1.83×10^(2) CFU/mL(纯菌液)。用人工污染牛奶样品验证RAA-LFD方法的可靠性,结果显示在增菌6 h时,RAA-LFD方法可检测到1.83×10^(1) CFU/mL金黄色葡萄球菌。分别采用微生物生化鉴定法、PCR法与RAA-LFD方法对64份牛奶样品进行金黄色葡萄球菌检测,结果表明,RAA-LFD方法与微生物生化鉴定法总符合率为95.3%,与PCR法总符合率(98.4%)表现出较高的一致性。本研究建立的可视化检测方法具有特异性强、灵敏度高、快速高效、操作简单、检测结果直观、对仪器设备要求低等特点,为牛奶中金黄色葡萄球菌的检测提供新的发展方向。

In this study, our aim was to establish a sensitive and rapid approach for the visual and point-of-care detection of Staphylococcus aureus using recombinase-aided amplification(RAA) and lateral flow dipstick(LFD). We designed and screened three pairs of specific primers targeting conserved sequences in the nuc gene of S. aureus and optimized reaction conditions. The RAA-LFD assay took 20 min to amplify the target gene with a 200 nmol/L primer concentration at 33 ℃.The specificity analysis exhibited no cross-reaction with other pathogens. The sensitivity of the RAA-LFD method was as low as 4 fg/μL for DNA and 1.83 × 10^(2) CFU/m L for pure cultures. The reliability was verified using artificially contaminated milk samples, showing that the limit of detection(LOD) was 1.83 × 10^(1) CFU/mL after 6 h enrichment. When 64 milk samples were tested for S. aureus using the traditional culture method, polymerase chain reaction(PCR) and the RAA-LFD method, the results of RAA-LFD showed a high total coincidence rate of 95.3% and 98.4% with those of the two former methods respectively. The visual detection method established in this study has the characteristics of high specificity, high sensitivity, rapidity, simple operation, and low requirements for equipment, and can provide a new development direction for the detection of S. aureus in milk.