Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani
Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani
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DOI：
10.1016/j.mcp.2020.101622
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发表时间：
2020-10-01
影响因子：
3.3
通讯作者：
Dzantiev, Boris B.
中科院分区：
文献类型：
--
作者：
Ivanov, Aleksandr, V;Safenkova, Irina, V;Dzantiev, Boris B.
马铃薯茄病菌是最重要的细菌病原体之一，它感染马铃薯植物，造成严重的经济损失。本研究建立了一种侧流检测技术（LFA）结合等温DNA扩增技术，用于马铃薯黑胫病的快速、特异、敏感诊断。索拉尼为此目的选择了内切酶聚合酶扩增（RPA）。5对特异性引物分别对D.设计并筛选了Solani基因组。将提供靶序列正确识别的引物对与对D. solani，侧翼为荧光素（正向引物）和生物素（反向引物）。构建侧向流测试条以检测DNA扩增子。RPA-LFA的检测限为14，000 D。每克马铃薯块茎的solani菌落形成单位。该试验提供了相应的聚合酶链反应（PCR）的灵敏度，但在固定的温度（39 ℃）超过30分钟。没有观察到非特异性反应与果胶杆菌，棒杆菌，和其他Dickeya物种。检测D.通过PCR验证了RPA-LFA对马铃薯块茎中solani的特异性。所获得的结果证实，RPA-LFA具有很大的潜力，用于高灵敏度检测潜伏感染。
Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. In this study, a lateral flow assay (LFA) combined with isothermal DNA amplification was developed for rapid, specific, and sensitive diagnosis of the potato blackleg disease caused by D. solani. Recombinase polymerase amplification (RPA) was chosen for this purpose. Five primer pairs specific to different regions of the D. solani genome were designed and screened. A primer pair providing correct recognition of the target sequence was aligned with the SOL-C region specific to D. solani and flanked by fluorescein (forward primer) and biotin (reverse primer). Lateral flow test strips were constructed to detect DNA amplicons. The RPA-LFA demonstrated a detection limit equal to 14,000 D. solani colony-forming units per gram of potato tuber. This assay provided sensitivity corresponding to the polymerase chain reaction (PCR) but was implemented at a fixed temperature (39 degrees C) over 30 min. No unspecific reactions with Pectobacterium, Clavibacter, and other Dickeya species were observed. Detection of latent infection of D. solani in the potato tubers by the developed RPA-LFA was verified by PCR. The obtained results confirmed that RPA-LFA has great potential for highly sensitive detection of latent infection.