Wageningen UR Food & Biobased Research, Biomolecular Sensing & Diagnostics, P.O. Box 17, 6700 AA, Wageningen, the Netherlands.
Anal Bioanal Chem. 2011 Jan;399(2):831-8. doi: 10.1007/s00216-010-4334-z. Epub 2010 Oct 28.
The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 10(4) and 10(5) colony forming units/mL or 0.1-0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.
基于侧向流条（核酸侧向流免疫分析，NALFIA）的使用，展示了碳纳米粒子在检测和鉴定不同产志贺毒素大肠杆菌毒力因子（vt1、vt2、eae 和 ehxA）和 16S 对照（特异性大肠杆菌）中的应用。在进行 NALFIA 检测之前，应用了带有标记引物的快速扩增方法。在优化的 NALFIA 条带的评估中，未发现任何抗体发生交叉反应。检测限高于定量 PCR（q-PCR），在大多数情况下，介于 10（4）和 10（5）个菌落形成单位/mL 或 0.1-0.9ng/μL DNA 之间。NALFIA 条带应用于来自牛粪便的 48 个分离株，并将结果与 q-PCR 进行比较。NALFIA 鉴定的大肠杆菌毒力因子与 q-PCR 观察到的非常吻合，在大多数情况下，灵敏度和特异性值均为 1.0，两种方法之间具有近乎完美的一致性（kappa 系数大于 0.9）。结果表明，所开发的筛选方法可靠、具有成本效益且易于使用，并且该过程快速，总耗时<1 小时，包括扩增。
