Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus.
Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus.
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DOI：
10.3389/fmicb.2017.00192
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发表时间：
2017
影响因子：
5.2
通讯作者：
Ye C
中科院分区：
文献类型：
--
作者：
Wang Y;Li H;Wang Y;Zhang L;Xu J;Ye C
该报告描述了一种简单，快速和灵敏的检测方法，用于视觉和多重检测粪肠球菌和金黄色葡萄球菌的基础上多环介导等温扩增（mLAMP）和侧流生物传感器（LFB）。Ef 0027基因（E.粪菌特异性基因）和nuc基因（S.金黄色葡萄球菌特异性基因）的表达。在存在生物素和FITC/地高辛修饰的引物的情况下，mLAMP产生大量生物素和FITC/地高辛连接的双链体产物，其通过生物素/链霉亲和素相互作用（双链体上的生物素和金纳米颗粒上的链霉亲和素）和免疫反应（双链体上的FITC/地高辛和LFB测试线上的抗FITC/地高辛）由LFB检测。金纳米颗粒的积累产生了一条特征性的红线，使目标病原体的视觉和多重检测无需仪器。在纯培养物和血液样品中成功地检测了LAMP-LFB技术的检测限（LoD）、分析特异性和可行性。该方法可在75 min内完成标本处理（30 min）、恒温反应（40 min）和结果报告（2 min），为大肠杆菌的多重检测提供了一种简便、快速、灵敏和特异的方法。faecalis和S.金黄色葡萄球菌此外，LAMP-LFB策略是一种通用的技术，通过重新设计特异性LAMP引物，可以将其扩展到检测各种靶序列。
The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.